Crystal structures and methods using same

ABSTRACT

The present invention relates generally to the fields of molecular biology and growth factor regulation. More specifically, the invention concerns modulators of FGFR3 function, and the identification and uses of said modulators.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.12/661,852, filed Mar. 24, 2010 which claims priority to U.S. patentapplication No. 61/163,222, filed on Mar. 25, 2009, the contents ofwhich are incorporated herein by reference.

REFERENCE TO TABLE SUBMITTED

This application is accompanied by a Table submitted in .txt formatwhich contains the file titled “P4294R1-1C1 Table 6.txt”, the contentsof which are incorporated in their entirety herein by reference.

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20120321606A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

FIELD OF THE INVENTION

The present invention relates generally to the field of molecularbiology. More specifically, the invention concerns anti-FGFR3antibodies, and uses of same.

BACKGROUND OF THE INVENTION

Fibroblast growth factors (FGFs) and their receptors (FGFRs) playcritical roles during embryonic development, tissue homeostasis andmetabolism (1-3). In humans, there are 22 FGFs (FGF1-14, FGF16-23) andfour FGF receptors with tyrosine kinase domain (FGFR1-4). FGFRs consistof an extracellular ligand binding region, with two or threeimmunoglobulin-like domains (IgD1-3), a single-pass transmembraneregion, and a cytoplasmic, split tyrosine kinase domain. FGFR1, 2 and 3each have two major alternatively spliced isoforms, designated IIIb andMc. These isoforms differ by about 50 amino acids in the second half ofIgD3, and have distinct tissue distribution and ligand specificity. Ingeneral, the Mb isoform is found in epithelial cells, whereas IIIc isexpressed in mesenchymal cells. Upon binding FGF in concert with heparansulfate proteoglycans, FGFRs dimerize and become phosphorylated atspecific tyrosine residues. This facilitates the recruitment of criticaladaptor proteins, such as FGFR substrate 2 α (FRS2α), leading toactivation of multiple signaling cascades, including themitogen-activated protein kinase (MAPK) and PI3K-AKT pathways (1, 3, 4).Consequently, FGFs and their cognate receptors regulate a broad array ofcellular processes, including proliferation, differentiation, migrationand survival, in a context-dependent manner.

Aberrantly activated FGFRs have been implicated in specific humanmalignancies (1, 5). In particular, the t(4; 14) (p16.3; q32)chromosomal translocation occurs in about 15-20% of multiple myelomapatients, leading to overexpression of FGFR3 and correlates with shorteroverall survival (6-9). FGFR3 is implicated also in conferringchemoresistance to myeloma cell lines in culture (10), consistent withthe poor clinical response of t(4; 14)+ patients to conventionalchemotherapy (8). Overexpression of mutationally activated FGFR3 issufficient to induce oncogenic transformation in hematopoietic cells andfibroblasts (11-14, 15), transgenic mouse models (16), and murine bonemarrow transplantation models (16, 17). Accordingly, FGFR3 has beenproposed as a potential therapeutic target in multiple myeloma. Indeed,several small-molecule inhibitors targeting FGFRs, although notselective for FGFR3 and having cross-inhibitory activity toward certainother kinases, have demonstrated cytotoxicity against FGFR3-positivemyeloma cells in culture and in mouse models (18-22).

FGFR3 overexpression has been documented also in a high fraction ofbladder cancers (23, 24). Furthermore, somatic activating mutations inFGFR3 have been identified in 60-70% of papillary and 16-20% ofmuscle-invasive bladder carcinomas (24, 25). In cell cultureexperiments, RNA interference (11, 26) or an FGFR3 single-chain Fvantibody fragment inhibited bladder cancer cell proliferation (27). Arecent study demonstrated that an FGFR3 antibody-toxin conjugateattenuates xenograft growth of a bladder cancer cell line throughFGFR3-mediated toxin delivery into tumors (28). However, it remainsunclear whether FGFR3 signaling is indeed an oncogenic driver of in vivogrowth of bladder tumors. Moreover, the therapeutic potential fortargeting FGFR3 in bladder cancer has not been defined on the basis ofin vivo models. Publications relating to FGFR3 and anti-FGFR3 antibodiesinclude co-pending, co-owned U.S. patent application Ser. No. ______(attorney docket P4294R1) filed Mar. 24, 2010, U.S. Patent Publicationno. 2005/0147612; Rauchenberger et al, J Biol Chem 278 (40):38194-38205(2003); WO2006/048877; Martinez-Torrecuadrada et al, (2008) Mol CancerTher 7(4): 862-873; WO2007/144893; Trudel et al. (2006) 107(10):4039-4046; Martinez-Torrecuadrada et al (2005) Clin Cancer Res 11 (17):6280-6290; Gomez-Roman et al (2005) Clin Cancer Res 11:459-465; Direnzo,R et al (2007) Proceedings of AACR Annual Meeting, Abstract No. 2080;WO2010/002862.

It is clear that there continues to be a need for agents that haveclinical attributes that are optimal for development as therapeuticagents. The invention described herein meets this need and providesother benefits.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a crystal comprising an anti-FGFR3antibody and an FGFR3, the crystal having, for example, the structuralcoordinates of Table 6. In one aspect, the invention provides a heavyatom derivative of a crystal of the invention. In one aspect, theinvention provides a composition comprising a crystal of the invention.

In one aspect, the invention provides a computer-implemented method, acomputer system and machine-readable data storage medium comprising adata storage material encoded with machine-readable instructions forcausing a display of a graphical three-dimensional representation of astructure of a portion of a crystal of anti-FGFR3 antibody (orstructural homolog thereof) in complex with FGFR3 (or a structuralhomolog and/or portion thereof). In some embodiments, the computer isprogrammed with instructions for transforming the structure coordinatesinto the graphical three-dimensional representation of the structureand/or displaying the graphical three-dimensional representation. Insome embodiments, the structure coordinates include the coordinates ofthe backbone atoms of the portion of the crystal and/or one or more ofthe contact residues between the anti-FGFR3 antibody and the FGFR3 inthe complex (e.g., some or all of the coordinates shown in Table 6).

In one aspect, the amino acid residues that form a binding site for aninhibitor binding site on FGFR3 are identified and are useful, forexample, in methods to model the structure of an FGFR3 binding site andto identify agents that can bind or fit into the binding site. This useincludes the rational design of modulators of FGFR3 activity. Forexample, these modulators include ligands that interact with FGFR3 andmodulate FGFR/FGF activities.

In some embodiments, the crystals are formed from an FGFR3 sequencecomprising sequence

ADPDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKSWISESVEADVRLRLANVSERDGGEYLCRATNFIGVAEKAFWLSVHGPRAAEEELVEA DEAGSV (SEQID NO:272) and an anti-FGFR3 antibody.

In some embodiments, the crystals are formed from an FGFR3 sequencecomprising sequence

ADPDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKSWISESVEADVRLRLANVSERDGGEYLCRATNFIGVAEKAFWLSVHGPRAAEEELVEA DEAGSVHHHHHH(SEQ ID NO:273) and an anti-FGFR3 antibody.

In some embodiments, the anti-FGFR3 antibody comprises a light chainvariable region comprising HVR-L1, HVR-L2, HVR-L3, wherein each, inorder, comprises SEQ ID NO:4, 5, 6, and/or a heavy chain variable regioncomprising HVR-H1, HVR-H2, and HVR-H3, where each, in order, containsSEQ ID NO: 1, 2, 3. In some embodiments, the anti-FGFR3 antibodycomprises a light chain variable region comprising sequence

(SEQ ID NO: 274) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQ GTKVEIKRand a heavy chain variable region comprising sequence

(SEQ ID NO: 275) EVQLVESGGGLVQPGGSLRLSCAASGFTFTSTGISWVRQAPGKGLEWVGRIY PTN GSTNYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARARTYGIYDLYVDYTEYVMDYWGQGTLV.

In some embodiments, the anti-FGFR3 antibody comprises:

(a) at least one, two, three, four, or five hypervariable region (HVR)sequences selected from:

(i) HVR-L1 comprising sequence A1-A11, wherein A1-A11 is RASQDVDTSLA(SEQ ID NO:87),

(ii) HVR-L2 comprising sequence B1-B7, wherein B1-B7 is SASFLYS (SEQ IDNO:88),

(iii) HVR-L3 comprising sequence C1-C9, wherein C1-C9 is QQSTGHPQT (SEQID NO:89),

(iv)) HVR-H1 comprising sequence D1-D10, wherein D1-D10 is GFTFTSTGIS(SEQ ID NO:84),

(v) HVR-H2 comprising sequence E1-E18, wherein E1-E18 isGRIYPTSGSTNYADSVKG (SEQ ID NO:85), and

(vi) HVR-H3 comprising sequence F1-F20, wherein F1-F20 isARTYGIYDLYVDYTEYVMDY (SEQ ID NO:86); and

(b) at least one variant HVR, where the variant HVR sequence comprisesmodification of at least one residue (at least two residues, at leastthree or more residues) of the sequence depicted in SEQ ID NOS:1-18,48-131 and 140-145. The modification desirably is a substitution,insertion, or deletion.

In some embodiments, a HVR-L1 variant comprises 1-6 (1, 2, 3, 4, 5, or6) substitutions in any combination of the following positions: A5 (V orD), A6 (V or I), A7 (D, E or S), A8 (T or I), A9 (A or S) and A10 (V orL). In some embodiments, a HVR-L2 variant comprises 1-2 (1 or 2)substitutions in any combination of the following positions: B1 (S orG), B4 (F or S or T) and B6 (A or Y). In some embodiments, a HVR-L3variant comprises 1-6 (1, 2, 3, 4, 5, or 6) substitutions in anycombination of the following positions: C3 (G or S or T), C4 (T or Y orA), C5 (G or S or T or A), C6 (A or H or D or T or N), C7 (Q or P or S),and C8 (S or Y or L or P or Q). In some embodiment, a HVR-H1 variantcomprises 1-3 (1, 2, or 3) substitutions in any combination of thefollowing positions: D3 (S or T), D5 (W or Y or S or T), D6 (S or G orT). In some embodiment, a HVR-H2 variant comprises 1-6 (1, 2, 3, 4, 5,or 6) substitutions in any combination of the following positions: E2 (Ror S), E6 (Y or A or L or S or T), E7 (A or Q or D or G or Y or S or Nor F), E8 (A or D or G), E9 (T or S), E10 (K or F or T or S), E11 (Y orH or N or I).

In some embodiments, the anti-FGFR3 antibody comprises:

(a) at least one, two, three, four, or five hypervariable region (HVR)sequences selected from:

(i) HVR-L1 comprising sequence RASQX₁X₂X₃X₄X₅X₆A, wherein X₁ is V or D,X₂ is V or I, X₃ is D, E or S, X₄ is T or I, X₅ is A or S, and X₆ is Vor L (SEQ ID NO:146),

(ii) HVR-L2 comprising sequence X₁ASFLX₂S wherein X₁ is S or G and X₂ isA or Y (SEQ ID NO:147),

(iii) HVR-L3 comprising sequence QQX₁X₂X₃X₄X₅X₆T, wherein X₁ is G, S orT, X₂ is T, Y or A, X₃ is G, S, T, or A, X₄ is A, H, D, T, or N, X₅ isQ, P or S, X₆ is S, Y, L, P or Q (SEQ ID NO:148),

(iv)) HVR-H1 comprising sequence GFX₁FX₂X₃TGIS, wherein X₁ is S or T, X₂is W, Y, S or T, X₃ is S, G, or T (SEQ ID NO:149),

(v) HVR-H2 comprising sequence GRIYPX₁X₂X₃X₄X₅X₆YADSVKG, wherein X₁ isY, A, L, 5, or T, X₂ is A, Q, D, G, Y, 5, N or F, X₃ is A, D, or G, X₄is T or 5, X₅ is K, F, T, or S, X₆ is Y, H, N or I (SEQ ID NO:150), and

(vi) HVR-H3 comprising sequence ARTYGIYDLYVDYTEYVMDY (SEQ ID NO:151).

In some embodiments, HVR-L1 comprises sequence RASQX₁VX₂X₃X₄VA, whereinX₁ is V or D, X₂ is D, E or S, X₃ is T or I, X₄ is A or S (SEQ IDNO:152). In some embodiments, HVR-L3 comprises sequence QQX₁X₂X₃X₄X₅X₆T,wherein X₁ is S, G, or T, X₂ is Y, T, or A, X₃ is T or G, X₄ is T, H orN, X₅ is P or S, X₆ is P, Q, Y, or L (SEQ ID NO:153). In someembodiments, HVR-H2 comprises sequence GRIYPX₁X₂GSTX₃YADSVKG, wherein X₁is T or L, X₂ is N, Y, S, G, A, or Q; X₃ is N or H (SEQ ID NO:154).

In one aspect, an anti-FGFR3 antibody comprises a heavy chain variableregion comprising HVR-H1, HVR-H2, HVR-H3, wherein each, in order,comprises SEQ ID NO:1, 2, 3, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, containsSEQ ID NO: 4, 5, 6.

In another aspect, an-anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, wherein each, inorder, comprises SEQ ID NO:7, 8, 9, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO: 10, 11, 12.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO:13, 14, 15, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO:16, 17, 18.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO: 48, 49, 50, and/or a light chain variable regionHVR-L1, HVR-L2, and HVR-L3, where each, in order, comprises SEQ ID NO:51, 52, 53.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO: 54, 55, 56, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO: 57, 58, 59.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO:60, 61, 62, 63, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO: 63, 64, 65.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO:66, 67, 68, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO: 69, 70, 71.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO:72, 73, 74, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO: 75, 76, 77.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO:78, 79 80, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO:81, 82, 83.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO: 84, 85, 86, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO:87, 88, 89.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO: 90, 91, 92, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO:93, 94, 95.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO: 96, 97, 98, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO: 99, 100, 101.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO: 102, 103, 104, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO: 105, 106, 107.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO:108, 109, 110, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO: 111, 112, 113.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO:114, 115, 116, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO:117, 118, 119.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO:120, 121, 122, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO: 123, 124, 125.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO:126, 127, 128, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO:129, 130, 131.

In another aspect, an anti-FGFR3 antibody comprises a heavy chainvariable region comprising HVR-H1, HVR-H2, HVR-H3, where each, in order,comprises SEQ ID NO:143, 144, 145, and/or a light chain variable regioncomprising HVR-L1, HVR-L2, and HVR-L3, where each, in order, comprisesSEQ ID NO:140, 141, 142.

The amino acid sequences of SEQ ID NOs:1-18, 48-131 and 140-145 arenumbered with respect to individual HVR (i.e., H1, H2 or H3) asindicated in FIG. 1, the numbering being consistent with the Kabatnumbering system as described below.

In some embodiments, the anti-FGFR3 antibody comprises a heavy chainvariable region comprising SEQ ID NO:132 and a light chain variableregion.

In some embodiments, the anti-FGFR3 antibody comprises a light chainvariable region comprising SEQ ID NO: 133, and a heavy chain variableregion.

In some embodiments, the anti-FGFR3 antibody comprises a heavy chainvariable region comprising SEQ ID NO:132 and a light chain variableregion comprising SEQ ID NO:133.

In some embodiments, the anti-FGFR3 antibody comprises a heavy chainvariable region comprising SEQ ID NO:134 and a light chain variableregion.

In some embodiments, the anti-FGFR3 antibody comprises a light chainvariable region comprising SEQ ID NO: 135, and a heavy chain variableregion.

In some embodiments, the anti-FGFR3 antibody comprises a light chainvariable region comprising SEQ ID NO: 139, and a heavy chain variableregion.

In some embodiments, the anti-FGFR3 antibody comprises a heavy chainvariable region comprising SEQ ID NO:134 and a light chain variableregion comprising SEQ ID NO:135.

In some embodiments, the anti-FGFR3 antibody comprises a heavy chainvariable region comprising SEQ ID NO:136 and a light chain variableregion.

In some embodiments, the anti-FGFR3 antibody comprises a light chainvariable region comprising SEQ ID NO: 137, and a heavy chain variableregion.

In some embodiments, the anti-FGFR3 antibody comprises a heavy chainvariable region comprising SEQ ID NO:136 and a light chain variableregion comprising SEQ ID NO:137.

In some embodiments, the anti-FGFR3 antibody comprises a heavy chainvariable region comprising SEQ ID NO:138 and a light chain variableregion.

In some embodiments, the anti-FGFR3 antibody comprises a light chainvariable region comprising SEQ ID NO: 139, and a heavy chain variableregion.

In some embodiments, the anti-FGFR3 antibody comprises a heavy chainvariable region comprising SEQ ID NO:138 and a light chain variableregion comprising SEQ ID NO:139.

In some embodiments, the anti-FGFR3 antibody comprises: at least one,two, three, four, five, and/or six hypervariable region (HVR) sequencesselected from the group consisting of:

(a) HVR-L1 comprising sequence SASSSVSYMH (SEQ ID NO:155), SASSSVSYMH(SEQ ID NO:156) or LASQTIGTWLA (SEQ ID NO:157),

(b) HVR-L2 comprising sequence TWIYDTSILAS (SEQ ID NO:158), RWIYDTSKLAS(SEQ ID NO:159), or LLIYAATSLAD (SEQ ID NO:160),

(c) HVR-L3 comprising sequence QQWTSNPLT (SEQ ID NO:161), QQWSSYPPT (SEQID NO:162), or QQLYSPPWT (SEQ ID NO:163),

(d) HVR-H1 comprising sequence GYSFTDYNMY (SEQ ID NO:164), GYVFTHYNMY(SEQ ID NO:165), or GYAFTSYNMY (SEQ ID NO:166),

(e) HVR-H2 comprising sequence WIGYIEPYNGGTSYNQKFKG (SEQ ID NO:167),WIGYIEPYNGGTSYNQKFKG (SEQ ID NO:168), or WIGYIDPYIGGTSYNQKFKG (SEQ IDNO:169), and

(f) HVR-H3 comprising sequence ASPNYYDSSPFAY (SEQ ID NO:170), ARGQGPDFDV(SEQ ID NO:171), or ARWGDYDVGAMDY (SEQ ID NO:172).

In some embodiments, the anti-FGFR3 antibody comprises: at least one,two, three, four, five, and/or six hypervariable region (HVR) sequencesselected from the group consisting of:

(a) HVR-L1 comprising sequence SASSSVSYMH (SEQ ID NO:155),

(b) HVR-L2 comprising sequence TWIYDTSILAS (SEQ ID NO:158),

(c) HVR-L3 comprising sequence QQWTSNPLT (SEQ ID NO:161),

(d) HVR-H1 comprising sequence GYSFTDYNMY (SEQ ID NO:164),

(e) HVR-H2 comprising sequence WIGYIEPYNGGTSYNQKFKG (SEQ ID NO:167), and

(f) HVR-H3 comprising sequence ASPNYYDSSPFAY (SEQ ID NO:170).

In some embodiments, the anti-FGFR3 antibody comprises: at least one,two, three, four, five, and/or six hypervariable region (HVR) sequencesselected from the group consisting of:

(a) HVR-L1 comprising sequence SASSSVSYMH (SEQ ID NO:156),

(b) HVR-L2 comprising sequence RWIYDTSKLAS (SEQ ID NO:159),

(c) HVR-L3 comprising sequence QQWSSYPPT (SEQ ID NO:162),

(d) HVR-H1 comprising sequence GYVFTHYNMY (SEQ ID NO:165),

(e) HVR-H2 comprising sequence WIGYIEPYNGGTSYNQKFKG (SEQ ID NO:168), and

(f) HVR-H3 comprising sequence ARGQGPDFDV (SEQ ID NO:171).

In some embodiments, the anti-FGFR3 antibody comprises: at least one,two, three, four, five, and/or six hypervariable region (HVR) sequencesselected from the group consisting of:

(a) HVR-L1 comprising sequence LASQTIGTWLA (SEQ ID NO:157),

(b) HVR-L2 comprising sequence LLIYAATSLAD (SEQ ID NO:160),

(c) HVR-L3 comprising sequence QQLYSPPWT (SEQ ID NO:163),

(d) HVR-H1 comprising sequence GYAFTSYNMY (SEQ ID NO:166),

(e) HVR-H2 comprising sequence WIGYIDPYIGGTSYNQKFKG (SEQ ID NO:169), and

(f) HVR-H3 comprising sequence ARWGDYDVGAMDY (SEQ ID NO:172).

In some embodiments, the anti-FGFR3 antibody comprises (a) a light chaincomprising (i) HVR-L1 comprising sequence SASSSVSYMH (SEQ ID NO:155);(ii) HVR-L2 comprising sequence TWIYDTSILAS (SEQ ID NO:158); and (iii)HVR-L3 comprising sequence QQWTSNPLT (SEQ ID NO:161); and/or (b) a heavychain comprising (i) HVR-H1 comprising sequence GYSFTDYNMY (SEQ IDNO:164); (ii) HVR-H2 comprising sequence WIGYIEPYNGGTSYNQKFKG (SEQ IDNO:167); and (iii) HVR-H3 comprising sequence ASPNYYDSSPFAY (SEQ IDNO:170).

In some embodiments, the anti-FGFR3 antibody comprises (a) a light chaincomprising (i) HVR-L1 comprising sequence SASSSVSYMH (SEQ ID NO:156);(ii) HVR-L2 comprising sequence RWIYDTSKLAS (SEQ ID NO:159); and (iii)HVR-L3 comprising sequence QQWSSYPPT (SEQ ID NO:162); and/or (b) a heavychain comprising (i) HVR-H1 comprising sequence GYVFTHYNMY (SEQ IDNO:165); (ii) HVR-H2 comprising sequence WIGYIEPYNGGTSYNQKFKG (SEQ IDNO:168); and (iii) HVR-H3 comprising sequence ARGQGPDFDV (SEQ IDNO:171).

In some embodiments, the anti-FGFR3 antibody comprises (a) a light chaincomprising (i) HVR-L1 comprising sequence LASQTIGTWLA (SEQ ID NO:157);(ii) HVR-L2 comprising sequence LLIYAATSLAD (SEQ ID NO:160); and (iii)HVR-L3 comprising sequence QQLYSPPWT (SEQ ID NO:163); and/or (b) a heavychain comprising (i) HVR-H1 comprising sequence GYAFTSYNMY (SEQ IDNO:166); (ii) HVR-H2 comprising sequence WIGYIDPYIGGTSYNQKFKG (SEQ IDNO:169); and (iii) HVR-H3 comprising sequence ARWGDYDVGAMDY (SEQ IDNO:172).

Some embodiments of antibodies of the invention comprise a light chainvariable domain of humanized 4D5 antibody (huMAb4D5-8) (HERCEPTIN®,Genentech, Inc., South San Francisco, Calif., USA) (also referred to inU.S. Pat. No. 6,407,213 and Lee et al., J. Mol. Biol. (2004),340(5):1073-1093) as depicted in SEQ ID NO:173 below.

(SEQ ID NO: 173) 1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu SerAla Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val 

 Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu IleTyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 

 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe AlaThr Tyr Tyr Cys Gln Gln 

 Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 107(HVR residues are underlined)In one embodiment, the huMAb4D5-8 light chain variable domain sequenceis modified at one or more of positions 30, 66, and 91 (Asn, Arg, andHis as indicated in bold/italics above, respectively). In a particularembodiment, the modified huMAb4D5-8 sequence comprises Ser in position30, Gly in position 66, and/or Ser in position 91. Accordingly, in oneembodiment, an antibody of the invention comprises a light chainvariable domain comprising the sequence depicted in SEQ ID NO:174 below:

(SEQ ID NO: 174) 1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu SerAla Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val 

 Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu IleTyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 

 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe AlaThr Tyr Tyr Cys Gln Gln 

 Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 107(HVR residues are underlined)Substituted residues with respect to huMAb4D5-8 are indicated inbold/italics.

Antibodies of the invention can comprise any suitable framework variabledomain sequence, provided binding activity to FGFR3 is substantiallyretained. For example, in some embodiments, antibodies of the inventioncomprise a human subgroup III heavy chain framework consensus sequence.In one embodiment of these antibodies, the framework consensus sequencecomprises a substitution at position 71, 73, and/or 78. In someembodiments of these antibodies, position 71 is A, 73 is T and/or 78 isA. In one embodiment, these antibodies comprise heavy chain variabledomain framework sequences of huMAb4D5-8 (HERCEPTIN®, Genentech, Inc.,South San Francisco, Calif., USA) (also referred to in U.S. Pat. Nos.6,407,213 & 5,821,337, and Lee et al., J. Mol. Biol. (2004),340(5):1073-1093). In one embodiment, these antibodies further comprisea human κI light chain framework consensus sequence. In a particularembodiment, these antibodies comprise light chain HVR sequences ofhuMAb4D5-8 as described in U.S. Pat. Nos. 6,407,213 & 5,821,337.) In oneembodiment, these antibodies comprise light chain variable domainsequences of huMAb4D5-8 (HERCEPTIN®, Genentech, Inc., South SanFrancisco, Calif., USA) (also referred to in U.S. Pat. Nos. 6,407,213 &5,821,337, and Lee et al., J. Mol. Biol. (2004), 340(5):1073-1093).

In one embodiment, an antibody of the invention comprises a heavy chainvariable domain, wherein the framework sequence comprises the sequenceof SEQ ID NOS:19 and 203-205, 20 and 206-208, 21 and 209-211, 22 and212-214, 23 and 215-217, 24 and 218-220, 25 and 221-223, 26 and 224-226,27 and 227-229, 28 and 230-232, 29 and 233-235, 30 and 236-238, 31 and239-241, 32 and 242-244, 33 and 245-247, 34 and 248-250, 35 and 251-253,36 and 254-256, and/or 37 and 257-259, and HVR H1, H2, and H3 sequencesare SEQ ID NOS:13, 14 and/or 15, respectively. In another embodiment,the framework sequence comprises the sequence of SEQ ID NOS: 19 and203-205, 20 and 206-208, 21 and 209-211, 22 and 212-214, 23 and 215-217,24 and 218-220, 25 and 221-223, 26 and 224-226, 27 and 227-229, 28 and230-232, 29 and 233-235, 30 and 236-238, 31 and 239-241, 32 and 242-244,33 and 245-247, 34 and 248-250, 35 and 251-253, 36 and 254-256, and/or37 and 257-259, and HVR H1, H2, and H3 sequences are SEQ ID NOS:48, 49and/or 50, respectively. In yet another embodiment, the frameworksequence comprises the sequence of SEQ ID NOS: 19 and 203-205, 20 and206-208, 21 and 209-211, 22 and 212-214, 23 and 215-217, 24 and 218-220,25 and 221-223, 26 and 224-226, 27 and 227-229, 28 and 230-232, 29 and233-235, 30 and 236-238, 31 and 239-241, 32 and 242-244, 33 and 245-247,34 and 248-250, 35 and 251-253, 36 and 254-256, and/or 37 and 257-259,and HVR H1, H2, and H3 sequences are SEQ ID NOS:84, 85, and/or 86,respectively. In a further embodiment, the framework sequence comprisesthe sequence of SEQ ID NOS: 19 and 203-205, 20 and 206-208, 21 and209-211, 22 and 212-214, 23 and 215-217, 24 and 218-220, 25 and 221-223,26 and 224-226, 27 and 227-229, 28 and 230-232, 29 and 233-235, 30 and236-238, 31 and 239-241, 32 and 242-244, 33 and 245-247, 34 and 248-250,35 and 251-253, 36 and 254-256, and/or 37 and 257-259, and HVR H1, H2,and H3 sequences are SEQ ID NOS:108, 109, and/or 110, respectively.

In a particular embodiment, an antibody of the invention comprises alight chain variable domain, wherein the framework sequence comprisesthe sequence of SEQ ID NOS:38 and 260-262, 39 and 263-265, 40 and266-268, and/or 41 and 269-271, and HVR L1, L2, and L3 sequences are SEQID NOS:16, 17, and/or 18, respectively. In another embodiment, anantibody of the invention comprises a light chain variable domain,wherein the framework sequence comprises the sequence of SEQ ID NOS: 38and 260-262, 39 and 263-265, 40 and 266-268, and/or 41 and 269-271, andHVR L1, L2, and L3 sequences are SEQ ID NOS:51, 52 and/or 53,respectively. In an additional embodiment, an antibody of the inventioncomprises a light chain variable domain, wherein the framework sequencecomprises the sequence of SEQ ID NOS: 38 and 260-262, 39 and 263-265, 40and 266-268, and/or 41 and 269-271, and HVR L1, L2, and L3 sequences areSEQ ID NOS:87, 88 and/or 89, respectively. In yet another embodiment, anantibody of the invention comprises a light chain variable domain,wherein the framework sequence comprises the sequence of SEQ ID NOS: 38and 260-262, 39 and 263-265, 40 and 266-268, and/or 41 and 269-271, andHVR L1, L2, and L3 sequences are SEQ ID NOS:111, 112, and/or 113,respectively.

In another aspect, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NO:132 and/or a lightchain variable domain comprising the sequence of SEQ ID NO:133. Inanother aspect, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NO:134 and/or a lightchain variable domain comprising the sequence of SEQ ID NO:135. Inanother aspect, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NO:136 and/or a lightchain variable domain comprising the sequence of SEQ ID NO:137. Inanother aspect, an antibody of the invention comprises a heavy chainvariable domain comprising the sequence of SEQ ID NO:138 and/or a lightchain variable domain comprising the sequence of SEQ ID NO:139.

In one aspect, the invention provides an anti-FGFR3 antibody that bindsa polypeptide comprising, consisting essentially of or consisting of thefollowing amino acid sequence: LAVPAANTVRFRCPA (SEQ ID NO:179) and/orSDVEFHCKVYSDAQP (SEQ ID NO:180).

In some embodiments, the antibody binds a polypeptide comprising,consisting essentially of or consisting of amino acid numbers 164-178and/or 269-283 of human FGFR3.

In one embodiment, an anti-FGFR3 antibody of the invention specificallybinds an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%,95%, 98% sequence identity or similarity with the sequenceLAVPAANTVRFRCPA (SEQ ID NO:179) and/or SDVEFHCKVYSDAQP (SEQ ID NO:180).

In one aspect, the anti-FGFR3 antibody of the present invention binds toat least one, two, three, four, or any number up to all of residues 154,155, 158, 159, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 177, 202, 205, 207, 210, 212, 214, 216, 217, 241,246, 247, 248, 278, 279, 280, 281, 282, 283, 314, 315, 316, 317 and/or318 of FGFR3-IIIb polypeptide, or equivalent residues of FGFR3-IIIcpolypeptide. One of ordinary skill in the art understands how to alignFGFR3 sequences in order identify corresponding residues betweenrespective FGFR3 sequences. Combinations of two or more residues caninclude any of residues 154, 155, 158, 159, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 177, 202, 205, 207,210, 212, 214, 216, 217, 241, 246, 247, 248, 278, 279, 280, 281, 282,283, 314, 315, 316, 317 and/or 318, or mixtures thereof, of FGFR3-IIIbpolypeptide, or equivalent residues of FGFR3-IIIc polypeptide. In someembodiments, the anti-FGFR3 antibody binds to at least one, two, three,four, or any number up to all of residues 158, 159, 169, 170, 171, 173,175, 205, 207, and/or 315, or mixtures thereof, of FGFR3-IIIbpolypeptide, or equivalent residues of FGFR3-IIIc polypeptide. In someembodiments, the anti-FGFR3 antibody binds to at least one, two three,four, or any number up to all of residues 158, 170, 171, 173, 175,and/or 315, or mixtures thereof, of FGFR3-IIIb polypeptide, orequivalent residues of FGFR3-IIIc polypeptide.

In one aspect, the invention provides methods of screening for acandidate inhibitor substance that inhibits FGFR3 activity, said methodcomprising: detecting binding, if any, of the candidate substance to aFGFR3 binding site, wherein the candidate substance is identified by amethod comprising comparing amount of FGFR3 activity in a sample withamount of FGFR3 activity in a reference sample comprising similaramounts of FGFR3 as the first sample but that has not been contactedwith said candidate substance, whereby a decrease in amount of FGFR3activity in the first sample compared to the reference sample indicatesthat the candidate substance is capable of inhibiting FGFR3 activity.

In another aspect, the invention provides methods of screening for acandidate inhibitory substance that inhibits FGFR3 activation, themethod comprising screening for a candidate inhibitory substance thatbinds FGFR3 binding site and inhibits FGFR3 activity.

In some embodiments, the methods comprising selecting for a substancethat binds to at least one, two, three, four, or any number up to all ofresidues 158, 170, 171, 173, 175, and/or 315 of the sequence ofFGFR3-IIIb polypeptide, or equivalent residues of FGFR3-IIIcpolypeptide. In some embodiments, the methods comprising selecting for asubstance that binds to at least one, two, three, four, or any number upto all of residues 154, 155, 158, 159, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, 175, 177, 202, 205, 207, 210,212, 214, 216, 217, 241, 246, 247, 248, 278, 279, 280, 281, 282, 283,314, 315, 316, 317, 318 of the sequence of FGFR3-IIIb polypeptide, orequivalent residues of FGFR3-IIIc polypeptide.

In some embodiments, the sample comprises FGFR3 and FGFR3 ligand (suchas FGF1 or FGF9). In some embodiments, the sample comprises a mammaliancell expressing FGFR3. In some embodiments, the FGFR3 is transgenicallyexpressed. In some embodiments, the sample comprises a Ba/FC cellexpressing FGFR3.

In some embodiments, FGFR3 activity comprises FGF (such as FGF1 and/orFGF9) binding, FGFR3 downstream molecular signaling, FGFR3phosphorylation, FGFR3 binding to a ligand (e.g., FGF1, FGF9), FGFR3dimerization, promotion of formation of monomeric FGFR3, and/ortreatment and/or prevention of a tumor, cell proliferative disorder or acancer; and/or treatment or prevention of a disorder associated withFGFR3 expression and/or activity (such as increased FGFR3 expressionand/or activity). In some embodiments, decrease in amount of FGFR3activity is reduction or blocking of FGF (such as FGF1 and/or FGF9)binding to FGFR3, reduction or blocking of FGFR3 activation, reductionor blocking of FGFR3 downstream molecular signaling), reduction orblocking of FGFR3 dimerization and/or treatment and/or prevention of atumor, cell proliferative disorder or a cancer; and/or treatment orprevention of a disorder associated with FGFR3 expression and/oractivity (such as increased FGFR3 expression and/or activity).

The invention also provides an antagonist molecule that inhibits FGFR3,wherein the molecule binds to at least one, two, three, four, or anynumber up to all of residues 158, 170, 171, 173, 175, and/or 315 of thesequence of FGFR3-IIIb polypeptide, or equivalent residues of FGFR3-IIIcpolypeptide. In some embodiments, the antagonist molecule binds to atleast one, two, three, four, or any number up to all of residues 154,155, 158, 159, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 177, 202, 205, 207, 210, 212, 214, 216, 217, 241,246, 247, 248, 278, 279, 280, 281, 282, 283, 314, 315, 316, 317, 318 ofthe sequence of FGFR3-IIIb polypeptide, or equivalent residues ofFGFR3-IIIc polypeptide. In some embodiments, the antagonist moleculecomprises an antibody.

The invention also provides methods using the antagonist molecules,including treatment and diagnostic methods.

In another aspect, the disclosure includes FGFR3 polypeptides andpolynucleotides encoding the polypeptides. The disclosure includes apolynucleotide encoding a polypeptide and/or a polypeptide having atleast 90% sequence identity to the polypeptide comprising sequence ofamino acids 154-318 of human FGFR3, not including the polypeptide havingthe amino acid sequence of human FGFR3. In some embodiments, thedisclosure includes a polynucleotide encoding a polypeptide and/or apolypeptide having at least 90% sequence identity to the polypeptidecomprising any of amino acid residue 154 to amino acid residue 177,amino acid residue 202 to amino acid reside 217, amino acid residue 241to amino acid residue 248, amino acid residue 278 to amino acid residue283 and/or amino acid residue 314 to amino acid residue 318 FGFR3, notincluding the polypeptide comprising the amino acid sequence of FGFR3.In some embodiments, the polypeptide binds a human FGFR3 ligand (such asFGF1 or FGF9).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C: Heavy chain and light chain HVR loop sequences ofanti-FGFR3 antibodies. The figures show the heavy chain HVR sequences,H1, H2, and H3, and light chain HVR sequences, L1, L2, and L3. Sequencenumbering is as follows:

Clone 184.6 (HVR-H1 is SEQ ID NO:1; HVR-H2 is SEQ ID NO:2; HVR-H3 is SEQID NO:3; HVR-L1 is SEQ ID NO:4; HVR-L2 is SEQ ID NO:5; HVR-L3 is SEQ IDNO:6);

Clone 184.6.1 (HVR-H1 is SEQ ID NO:7; HVR-H2 is SEQ ID NO:8; HVR-H3 isSEQ ID NO:9; HVR-L1 is SEQ ID NO:10; HVR-L2 is SEQ ID NO:11; HVR-L3 isSEQ ID NO:12)

Clone 184.6.58 (HVR-H1 is SEQ ID NO:13; HVR-H2 is SEQ ID NO:14; HVR-H3is SEQ ID NO:15; HVR-L1 is SEQ ID NO:16; HVR-L2 is SEQ ID NO:17; HVR-L3is SEQ ID NO:18)

Clone 184.6.62 (HVR-H1 is SEQ ID NO:48; HVR-H2 is SEQ ID NO:49; HVR-H3is SEQ ID NO:50; HVR-L1 is SEQ ID NO:51; HVR-L2 is SEQ ID NO:52; HVR-L3is SEQ ID NO:53)

Clone 184.6.21 (HVR-H1 is SEQ ID NO:54; HVR-H2 is SEQ ID NO:55; HVR-H3is SEQ ID NO:56; HVR-L1 is SEQ ID NO:57; HVR-L2 is SEQ ID NO:58; HVR-L3is SEQ ID NO:59)

Clone 184.6.49 (HVR-H1 is SEQ ID NO:60; HVR-H2 is SEQ ID NO:61; HVR-H3is SEQ ID NO:62; HVR-L1 is SEQ ID NO:63; HVR-L2 is SEQ ID NO:64; HVR-L3is SEQ ID NO:65)

Clone 184.6.51 (HVR-H1 is SEQ ID NO:66; HVR-H2 is SEQ ID NO:67; HVR-H3is SEQ ID NO:68; HVR-L1 is SEQ ID NO:69; HVR-L2 is SEQ ID NO:70; HVR-L3is SEQ ID NO:71)

Clone 184.6.52 (HVR-H1 is SEQ ID NO:72; HVR-H2 is SEQ ID NO:73; HVR-H3is SEQ ID NO:74; HVR-L1 is SEQ ID NO:75; HVR-L2 is SEQ ID NO:76; HVR-L3is SEQ ID NO:77)

Clone 184.6.92 (HVR-H1 is SEQ ID NO:78; HVR-H2 is SEQ ID NO:79; HVR-H3is SEQ ID NO:80; HVR-L1 is SEQ ID NO:81; HVR-L2 is SEQ ID NO:82; HVR-L3is SEQ ID NO:83)

Clone 184.6.1.N54S (HVR-H1 is SEQ ID NO:84; HVR-H2 is SEQ ID NO:85;HVR-H3 is SEQ ID NO:86; HVR-L1 is SEQ ID NO:87; HVR-L2 is SEQ ID NO:88;HVR-L3 is SEQ ID NO:89)

Clone 184.6.1.N54G (HVR-H1 is SEQ ID NO:90; HVR-H2 is SEQ ID NO:91;HVR-H3 is SEQ ID NO:92; HVR-L1 is SEQ ID NO:93; HVR-L2 is SEQ ID NO:94;HVR-L3 is SEQ ID NO:95)

Clone 184.6.1.N54A (HVR-H1 is SEQ ID NO:96; HVR-H2 is SEQ ID NO:97;HVR-H3 is SEQ ID NO:98; HVR-L1 is SEQ ID NO:99; HVR-L2 is SEQ ID NO:100;HVR-L3 is SEQ ID NO:101)

Clone 184.6.1.N54Q (HVR-H1 is SEQ ID NO:102; HVR-H2 is SEQ ID NO:103;HVR-H3 is SEQ ID NO:104; HVR-L1 is SEQ ID NO:105; HVR-L2 is SEQ IDNO:106; HVR-L3 is SEQ ID NO:107)

Clone 184.6.58.N54S (HVR-H1 is SEQ ID NO:108; HVR-H2 is SEQ ID NO:109;HVR-H3 is SEQ ID NO:110; HVR-L1 is SEQ ID NO:111; HVR-L2 is SEQ IDNO:112; HVR-L3 is SEQ ID NO:113)

Clone 184.6.58.N54G (HVR-H1 is SEQ ID NO:114; HVR-H2 is SEQ ID NO:115;HVR-H3 is SEQ ID NO:116; HVR-L1 is SEQ ID NO:117; HVR-L2 is SEQ IDNO:118; HVR-L3 is SEQ ID NO:119)

Clone 184.6.58.N54A (HVR-H1 is SEQ ID NO:120; HVR-H2 is SEQ ID NO:121;HVR-H3 is SEQ ID NO:122; HVR-L1 is SEQ ID NO:123; HVR-L2 is SEQ IDNO:124; HVR-L3 is SEQ ID NO:125)

Clone 184.6.58.N54Q (HVR-H1 is SEQ ID NO:126; HVR-H2 is SEQ ID NO:127;HVR-H3 is SEQ ID NO:128; HVR-L1 is SEQ ID NO:129; HVR-L2 is SEQ IDNO:130; HVR-L3 is SEQ ID NO:131).

Clone 184.6.1.NS D30E (HVR-H1 is SEQ ID NO:143; HVR-H2 is SEQ ID NO:144;HVR-H3 is SEQ ID NO:145; HVR-L1 is SEQ ID NO:140; HVR-L2 is SEQ IDNO:141; HVR-L3 is SEQ ID NO:142).

Amino acid positions are numbered according to the Kabat numberingsystem as described below.

FIGS. 2A and 2B: depict (A) the amino acid sequences of the heavy chainvariable regions and light chain variable regions of anti-FGFR3antibodies 184.6.1.N54S, 184.6.58, and 184.6.62; and (B) thehypervariable regions of anti-FGFR3 antibodies 1G6, 6G1, and 15B2.

FIGS. 3A, 3B, and 4: depict exemplary acceptor human consensus frameworksequences for use in practicing the instant invention with sequenceidentifiers as follows:

Variable Heavy (VH) Consensus Frameworks (FIG. 3A, 3B)

human VH subgroup I consensus framework minus Kabat CDRs (SEQ ID NOS:19and 203-205)human VH subgroup I consensus framework minus extended hypervariableregions (SEQ ID NOS:20 and 206-208, 21 and 209-211, 22 and 212-214)human VH subgroup II consensus framework minus Kabat CDRs (SEQ ID NOS:23and 215-217)human VH subgroup II consensus framework minus extended hypervariableregions (SEQ ID NOS:24 and 218-220, 25 and 221-223, 26 and 224-226)human VH subgroup II consensus framework minus extendedhuman VH subgroup III consensus framework minus Kabat CDRs (SEQ IDNOS:27 and 227-229)human VH subgroup III consensus framework minus extended hypervariableregions (SEQ ID NOS:28 and 230-232, 29 and 233-235, 30 and 236-238)human VH acceptor framework minus Kabat CDRs (SEQ ID NOS:31 and 239-241)human VH acceptor framework minus extended hypervariable regions (SEQ IDNOS:32 and 242-244, 33 and 2245-247)human VH acceptor 2 framework minus Kabat CDRs (SEQ ID NOS:34 and248-250)human VH acceptor 2 framework minus extended hypervariable regions (SEQID NOS:35 and 251-253, 36 and 254-256, 37 and 257-259)

Variable Light (VL) Consensus Frameworks (FIG. 4)

human VL kappa subgroup I consensus framework (SEQ ID NO:38 and 260-262)human VL kappa subgroup II consensus framework (SEQ ID NO:39 and263-265)human VL kappa subgroup III consensus framework (SEQ ID NO:40 and266-268)human VL kappa subgroup IV consensus framework (SEQ ID NO:41 and269-271)

FIG. 5: depicts framework region sequences of huMAb4D5-8 light (SEQ IDNOS:42-45) and heavy chains (SEQ ID NOS:46, 47, 175, 176). Numbers insuperscript/bold indicate amino acid positions according to Kabat.

FIG. 6: depicts modified/variant framework region sequences ofhuMAb4D5-8 light (SEQ ID NOS:42, 43, 177, 45) and heavy chains (SEQ IDNOS:46, 47, 178, and 176). Numbers in superscript/bold indicate aminoacid positions according to Kabat.

FIG. 7: FGFR3 knockdown in bladder cancer cell RT112 inhibitsproliferation and induces G1 cell cycle arrest in vitro, and suppressestumor growth in vivo. Three different FGFR3 shRNAs were cloned into aTet-inducible expression vector. RT112 cells stably expressing FGFR3shRNAs or a control shRNA were established with puromycin selection. (A)Representative blots showing FGFR3 expression in selected clones treatedwith or without doxycycline (Dox, 0, 0.1 and 1 μg/ml, left to right).(B) [³H]-thymidine incorporation by RT112 stable cells. RT112 stableclones were cultured with or without 1 μg/ml doxycycline for 3 daysprior to 16 hour-incubation with [³H]-thymidine (1 μCi per well). Countsof incorporated [³H]-thymidine were normalized to that from cellswithout doxycycline induction. Error bars represent SEM. (C) DNAfluorescence flow cytometry histograms of RT112 stable cells. RT112clones expressing control shRNA or FGFR3 shRNA4 were cultured with orwithout 1 μg/ml doxycycline for 72 hours, and the nuclei were stainedwith propidium iodide (PI). Similar results were obtained for FGFR3shRNA2 and 6 (FIG. 16). (D) The growth of RT112 cells expressing controlshRNA (n=9 per treatment group) or FGFR3 shRNA4 (n=11 per treatmentgroup) in mice. Mice were given 5% sucrose alone or supplemented with 1mg/ml doxycycline, and tumor size was measured twice a week. Error barsrepresent SEM. Similar results were obtained for FGFR3 shRNA2 and 6(FIG. 16). Lower panel: Expression of FGFR3 protein in tumor lysatesextracted from control shRNA or FGFR3 shRNA4 stable cell xenografttissues.

FIG. 8: R3Mab blocks FGF/FGFR3 interaction. (A) Selective binding ofhuman FGFR3 by R3Mab. Human FGFR1-4 Fc chimeric proteins wereimmobilized and incubated with increasing amount of R3Mab. Specificbinding was detected using an anti-human Fab antibody. (B-C) Blocking ofFGF1 binding to human FGFR3-IIIb (B) or IIIc (C) by R3Mab. Specificbinding was detected by using a biotinylated FGF1-specific polyclonalantibody. (D-E) Blocking of FGF9 binding to human FGFR3-IIIb (D) or Mc(E) by R3Mab. Specific binding was detected by using a biotinylatedFGF9-specific polyclonal antibody. Error bars represent standard errorof the mean (SEM) and are sometimes smaller than symbols.

FIG. 9: R3Mab inhibits Ba/F3 cell proliferation driven by wild type andmutated FGFR3. (A) Inhibitory effect of R3Mab on the viability of Ba/F3cells expressing wild type human FGFR3-IIIb. Cells were cultured inmedium without FGF1 (no FGF1), or in the presence of 10 ng/ml FGF1 plus10 μg/ml heparin alone (FGF1), or in combination with a control antibody(Control) or R3Mab. Cell viability was assessed with CellTiter-Glo(Promega) after 72 hr incubation with antibodies. (B) Inhibition ofFGFR3 and MAPK phosphorylation by R3Mab in Ba/F3-FGFR3-IIIb^(WT) stablecells. Cells were treated with 15 ng/ml FGF1 and 10 μg/ml heparin (+) orheparin alone (−) for 10 minutes, following pre-incubation with aControl Ab (Ctrl), decreasing amount of R3Mab (1, 0.2, 0.04 μg/mlrespectively) in PBS, or PBS alone (Mock) for 3 hours. Lysates wereimmunoblotted to assess phosphorylation of FGFR3 and p44/42 MAPK withantibodies to pFGFR^(Y653/654) and pMAPK^(Thr202/Tyr204) respectively.(C) Schematic representation of FGFR3 mutation hot spots and frequencyin bladder cancer (sequence numbering depicted is based on theFGFR3-IIIb isoform amino acid sequence) based on published data (32).TM, transmembrane domain; TK1 and TK2, tyrosine kinase domain 1 and 2.(D-H) Inhibitory effect of R3Mab on the viability of Ba/F3 cellsexpressing cancer-associated FGFR3 mutants. G372C is derived from Mcisoform, and the rest are derived from Mb isoform. Sequence numberingfor all mutants is based on the FGFR3-IIIb isoform amino acid sequence(including the G372C mutant, which would be numbered G370C based on theFGFR3-IIIc isoform amino acid sequence). Cell viability was assessedafter 72 hour incubation with antibodies as described in (A). Error barsrepresent SEM.

FIG. 10: Epitope mapping for R3Mab and crystal structure of the complexbetween R3Mab Fab fragment and IgD2-D3 of human FGFR3-IIIb. (A) Epitopedetermined by the binding of 13 peptides spanning IgD2-D3 of human FGFR3to R3Mab. Each biotinylated peptide was captured ontostreptavidin-coated microtiter well and incubated with R3Mab.Specifically bound R3Mab was detected using a goat anti-human IgGantibody. (B) Sequence alignment of human FGFR3 peptides 3(LAVPAANTVRFRCPA (SEQ ID NO:179) and 11 (SDVEFHCKVYSDAQP (SEQ ID NO:180)with extracellular segments of human FGFR1 (peptide 3: HAVPAAKTVKFKCPS(SEQ ID NO:181); peptide 11: SNVEFMCKVYSDPQP (SEQ ID NO:182)). FGFR1residues engaged in the primary FGF2-FGFR1 interaction, heparin binding,and receptor-receptor association are shown in bold, italics, andunderlined font, respectively. Functional assignment of FGFR1 residuesis based on Plotnikov et al (34). (C) Structure of R3Mab Fab (shown inribbon-helix, light chain grey, heavy chain black) in complex with humanFGFR IgD2-D3 (shown in molecular surface, white). Receptor residuesinvolved in ligand binding and dimerization are colored ingrey/crosshatched and dark grey respectively based on Plotnikov et al(34). (D) The close-up of the crystal structure shows that CDR-H3 and-H2 from the Fab constitute the major interaction sites with IgD2 andIgD3 of FGFR3. (E) Superposition of FGFR3-IIIc-FGF1 complex (PDB code1RY7) with FGFR3-IIIb-Fab complex. FGFR3-IIIc and FGF1 are colored ingrey and dark grey respectively. FGFR3-IIIb is shown in white and theFab is shown in light grey for light chain, dark grey for heavy chain.IgD2 was used as the anchor for superposition. Note the well-superposedIgD2 from both structures and the new conformation adopted by IgD3 ofFGFR3-IIIb when bound by R3Mab. (F) Another representation of thesuperposition of FGFR3-IIIc-FGF1 complex (PDB code 1RY7) withFGFR3-IIIb-Fab complex. FGFR3-IIIc and FGF1 are shown as molecularsurfaces that are grey/mesh texture and dark grey/dotted texture,respectively. FGFR3-IIIb is shown in white and the Fab is shown in greyfor light chain, black for heavy chain. IgD2 was used as the anchor forsuperposition. Note the well-superposed IgD2 from both structures andthe new conformation adopted by IgD3 of FGFR3-IIIb when bound by R3Mab.

FIG. 11: R3Mab inhibits proliferation, clonal growth and FGFR3 signalingin bladder cancer cells expressing wild type or mutated FGFR3^(S249C).(A) Inhibition of [³H]-thymidine incorporation by R3Mab in bladdercancer cell line RT112. Error bars represent SEM. (B) Blocking ofFGF1-activated FGFR3 signaling by R3Mab (15 μg/ml) in bladder cancercell line RT112 as compared to treatment medium alone (Mock) or acontrol antibody (Ctrl). Cell lysates were immunoprecipitated withanti-FGFR3 antibody and assessed for FGFR3 phosphorylation with ananti-phospho-tyrosine antibody (4G10). Lysates were immunoblotted todetect phosphorylation of AKT (pAKT^(S473)) and p44/42 MAPK(pMAPK^(Thr202/Tyr204)). (C) Inhibition of clonal growth by R3Mab (10μg/ml) in bladder cancer cell line UMUC-14 (harboring FGFR3^(S249C)) ascompared to treatment medium alone (Mock) or a control antibody (Ctrl).(D) Quantitation of the study in (C) reporting the number of colonieslarger than 120 μm in diameter per well from a replicate of 12 wells.Error bars represent SEM. P<3.4×10⁻⁹ versus Mock or Ctrl. (E) Inhibitionof FGFR3 phosphorylation in UMUC-14 cells by R3Mab (15 μg/ml). FGFR3phosphorylation was analyzed as in (B). Note constitutivephosphorylation of FGFR3 in this cell line.

FIG. 12: R3Mab decreases steady-state level of disulfide-linkedFGFR3^(S249C) dimer by driving the dimer-monomer equilibrium towardmonomeric state. (A) Effect of R3Mab on FGFR3^(S249C) dimer in UMUC-14cells. Cells were incubated with R3Mab (15 μg/ml) or a control antibody(Ctrl) for 3 hours, and whole cell lysates were analyzed by immunoblotunder non-reducing and reducing conditions. (B) Effect offree-sulfhydryl blocker DTNB on FGFR3^(S249C) dimer-monomer equilibriumin UMUC-14 cells. UMUC-14 cells were treated with increasingconcentration of DTNB for 3 hours, and cell lysates were analyzed as in(A). (C) Effect of R3Mab on purified recombinant FGFR3^(S249C) dimer invitro. FGFR3^(S249C) dimer composed of IgD2-D3 was purified throughsize-exclusion column, and incubated with PBS (Mock), a control antibody(Ctrl), or R3Mab at 37° C. Samples were collected at indicated time forimmunoblot analysis under non-reducing conditions. FGFR3 dimer-monomerwas detected using anti-FGFR3 hybridoma antibody 6G1 (A-C).

FIG. 13: R3Mab inhibits xenograft growth of bladder cancer cells andallograft growth of Ba/F3-FGFR3^(S249C) (A) Effect of R3Mab on thegrowth of pre-established RT112 bladder cancer xenografts compared withvehicle control. n=10 per group. (B) Inhibition of FGFR3 signaling inRT112 tumor tissues by R3Mab. In a separate experiment, RT112 xenografttumors that were treated with 15 mg/kg of a control antibody (Ctrl) orR3Mab for 48 hours or 72 hours were collected (n=3 per group),homogenized and analyzed for FRS2α and MAPK activation by immunoblot.(C) Effect of R3Mab on the growth of pre-established Ba/F3-FGFR3^(S249C)allografts. n=10 per group. (D) Effect of R3Mab on the growth ofpre-established UMUC-14 bladder cancer xenografts, n=10 per group. (E)Effect of R3Mab on FGFR3^(S249C) dimer and signaling in UMUC-14 tumortissues. UMUC-14 xenograft tumors that were treated with 30 mg/kg of acontrol antibody (Ctrl) or R3Mab for 24 hours or 72 hours were collected(n=3 per group), homogenized, and analyzed for FGFR3^(S249C)dimer-monomer as well as MAPK activation by immunoblot. FGFR3dimer-monomer was detected using an anti-FGFR3 rabbit polyclonalantibody sc9007 to avoid interference from mouse IgG in tumor lysates.Error bars represent SEM.

FIG. 14: ADCC contributes to the anti-tumor efficacy of R3Mab in t(4;14) positive multiple myeloma models. (A-B) Effect of R3Mab on thegrowth of pre-established OPM2 (A) and KMS11 (B) myeloma xenografts.n=10 per group. (C-F) Cytolysis of myeloma cell lines OPM2 (C) and KMS11(D), or bladder cancer cell lines RT112 (E) and UMUC-14 (F) induced byR3Mab in cell culture. Myeloma or bladder cancer cells were incubatedwith freshly isolated human PBMC in the presence of R3Mab or a controlantibody. Cytotoxicity was determined by measuring LDH released in thesupernatant. (G-H) Effect of R3Mab or its DANA mutant on the growth ofpre-established OPM2 (G) and KMS11 (H) myeloma xenografts. n=10 pergroup. Error bars represent SEM and are sometimes smaller than symbols.

FIG. 15: Knockdown of FGFR3 with siRNA inhibits cell proliferation ofbladder cancer cell lines. Six to seven different FGFR3 siRNAs and threenon-specific control siRNAs were designed and synthesized in Genentech.Bladder cancer cell lines RT112 (A), SW780 (B), RT4 (C) and UMUC-14 (D)were plated into 96-well plate (3000 cells per well) and allowed toattach overnight, and transiently transfected with 25 nM siRNA incomplex with RNAiMax (Invitrogen). 72 hr post-transfection,[³H]-thymidine (1 μCi per well) was added to the culture (A, C, and D)for another 16 hour incubation. Incorporated [³H]-thymidine wasquantitated with TopCount. Data were normalized to that from cellstransfected with RNAiMax alone (Mock). Error bars represent SEM. Lowerpanel: Representative blots showing FGFR3 expression in siRNAtransfected cells. (B) Cell viability was measured with CellTiter-Glo(Promega) 96 hours after transfection. Error bars represent SEM.

FIG. 16: FGFR3 knockdown in bladder cancer cell line RT112 induces G1cell cycle arrest in vitro, and suppresses tumor growth in vivo. Threedifferent FGFR3 RNAs were designed and cloned into a Tet-inducible shRNAexpression retroviral vector. RT112 stable clones expressing FGFR3shRNAs or control shRNA were established with puromycin selection. (A)DNA fluorescence flow cytometry histograms of propidium iodide(PI)-stained nuclei obtained from RT112 stable cells expressing FGFR3shRNA2 or shRNA6 following treatment with or without 1 μg/ml doxycyclinefor 72 hours. (B) The growth of RT112 stable cells expressing FGFR3shRNA2-4 (n=11 per treatment group) or FGFR3shRNA6-16 (n=10 pertreatment group) in nu/nu mice. Tumor bearing mice received 5% sucroseonly (solid circle) or 5% sucrose plus 1 mg/ml doxycycline (solidsquare), and tumors were measured with calipers twice a week. Error barsrepresent SEM.

FIG. 17: Effect of anti-FGFR3 hybridoma antibodies 16G, 6G1 and 15B2 onBa/F3 cell proliferation driven by wild type and mutated FGFR3.Anti-FGFR3 hybridoma antibodies were generated by immunizing BALB/c micewith human FGFR3-IIIb/Fc or human FGFR3-IIIc/Fc chimera. Fused hybridomacells were selected using hypoxanthin-aminopterin-thymidine selection inMedium D from the ClonaCell® hybridoma selection kit (StemCellTechnologies, Inc., Vancouver, BC, Canada). Hybridoma antibodies weresequentially screened for their ability to bind to FGFR3-IIIb andFGFR3-IIIc by ELISA and to recognize cell surface FGFR3 by FACS.Selected hybridomas were then cloned by limiting dilution. 16G, 6G1 and15B2 are clones used to assess the effect on the proliferation of Ba/F3cells expressing wild type or mutated FGFR3 similarly as described inFIG. 9A. Error bars represent SEM.

FIG. 18: Comparison of R3Mab epitopes determined by peptide mapping andcrystal structure analysis. (A) Epitope revealed by the structure of theR3Mab Fab fragment in complex with the extracellular IgD2-D3 segment ofhuman FGFR3. FGFR3 residues contacted by Fab heavy chain and light chainare colored in black and grey, respectively. (B) Location of peptides 3and 11 on FGFR3.

FIG. 19: R3Mab inhibits proliferation and FGFR3 signaling in bladdercancer cells containing wild type or mutated FGFR3^(S249C). (A)Inhibition of cell viability by R3Mab in bladder cancer cell line RT4.Cell viability was assessed with CellTiter-Glo (Promega) after 96 hrincubation with the antibody. Error bars represent SEM. (B) Blocking ofFGF1-activated FGFR3 signaling by R3Mab (15 ug/ml) in bladder cancercell line RT4. (C) Inhibition of [³H]-thymidine incorporation by R3Mabin bladder cancer cell line RCC-97-7 (containing FGFR3^(S249C)). Errorbars represent SEM. (D) Inhibition of FGFR3 phosphorylation in TCC-97-7cells by R3Mab (15 ug/ml). (E) Decrease of FGFR3^(S249C) dimer inTCC-97-7 cells after 3 hours incubation with R3Mab (15 ug/ml) comparedwith a control antibody (Ctrl).

FIG. 20: Effect of endocytosis inhibitors on the internalization ofR3Mab and FGFR3^(S249C) dimer in UMUC-14 cells. (A) Effect ofendocytosis inhibitors on the internalization of R3Mab. UMUC-14 cells,pre-treated with various endocytosis inhibitor or DMSO for 1 hour at 37°C., were incubated with R3Mab (15 ug/ml) for 3 hours at 37° C. to allowinternalization. A low pH wash was used to remove cell surface R3Mab tovisualize internalized antibody. Cells were fixed and stained with Alexa488-labeled anti-human IgG. Image was taken using confocal microscopy.(B) Effect of endocytosis inhibitors on FGFR3^(S249C) dimer in UMUC-14cells treated with R3Mab. UMUC-14 cells, pre-treated with variousendocytosis inhibitor or DMSO for 1 hour at 37° C., were incubated withmock (Lane 1), a control antibody (Lane 2), or R3Mab (15 ug/ml, Lane 3)for 3 hours at 37° C. Cell lysates were analyzed for FGFR3 protein undernon-reducing or reducing conditions by immunoblot. Note thatchlorpromazine (inhibitor of clathrin-mediated endocytosis) andgenistein (pan-inhibitor of endocytosis) blocked R3Mab internalization,but had no effect on R3Mab-induced decrease of FGFR3^(S249C) dimer.

FIG. 21: Detection sensitivity of different anti-FGFR3 antibodies towardmonomeric and dimeric FGFR3^(S249C) under non-reducing conditions.UMUC-14 cells were lysed after treatment with R3Mab (Lane 1), a controlIgG1 (Lane 2), or PBS (Lane 3) for 3 hours, and cell lysates weresubject to immunoblot analyses under reducing or non-reducingconditions. Note that 6G1 (murine hybridoma antibody generated atGenentech) detected both FGFR3^(S249C) dimer and monomer, whereas sc9007(rabbit polyclonal antibody, Santa Cruz Biotechnology) or sc13121(murine hybridoma antibody, Santa Cruz Biotechnology) preferentiallydetected the dimeric FGFR3^(S249C).

FIG. 22: Effect of R3Mab on the proliferation of t(4; 14)+ multiplemyeloma cells. (A) Inhibitory effect of R3Mab on [³H]-thymidineincorporation by UTMC-2 cells. UTMC-2 cells were grown in mediumcontaining R3Mab or a control antibody in the presence of 25 ng/ml FGF9and 5 ug/ml heparin or heparin alone (No FGF9). After 6 days incubation,[³H]-thymidine was added for 16 hr incubation. Data were normalized tothat from cells grown in the absence of FGF9 and antibody. (B-C) Effectof R3Mab on [³H]-thymidine incorporation by OPM2 (B) and KMS11 (C)cells. Cells grown in 1.5% FBS medium were treated with R3Mab or acontrol antibody for 6 days. Data were normalized to that from untreatedcells. Error bars represent SEM.

FIG. 23: Cell surface expression levels of FGFR3 in myeloma and bladdercancer cells. (A) Cell surface FGFR3 expression in myeloma cells andbladder cancer cells assessed by FACS analysis. Cells were stained withphycoerythin-conjugated mouse mAb against human FGFR3 (FAB766P, R&DSystems) or phycoerythin-conjugated isotype control mouse IgG1 (BDPharmingen). (B) Scatchard analysis of FGFR3 density in myeloma cellsand bladder cancer cells. R3Mab was radioiodinated, and incubated withcells in suspension with excess unlabeled antibody. After incubation atRT for 2 hours, cells were pelleted by centrifugation and washed twice.Specifically bound ¹²⁵I was determined. Receptor density and bindingaffinity (Kd) represent the mean from two binding experiments.

FIG. 24: Effect of R3Mab or its DANA mutant on xenograft growth ofbladder carcinoma cells. (A) Effect of R3Mab and its DANA mutant (50mg/kg each) on the growth of pre-established RT112 tumors. (B) Effect ofR3Mab and its DANA mutant (50 mg/kg each) on the growth ofpre-established UMUC-14 tumors. Error bars represent SEM.

DETAILED DESCRIPTION OF THE INVENTION General Techniques

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3rd. edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS INMOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (2003)); the seriesMETHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICALAPPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMALCELL CULTURE (R. I. Freshney, ed. (1987)).

DEFINITIONS

The term “binding site,” as used herein, refers to a region of amolecule or molecular complex that, as a result of its shape,distribution of electrostatic charge, presentation of hydrogen-bondacceptors or hydrogen-bond donors, and/or distribution of nonpolarregions, favorably associates with a ligand. Thus, a binding site mayinclude or consist of features such as cavities, surfaces, or interfacesbetween domains. Ligands that may associate with a binding site include,but are not limited to, cofactors, substrates, receptors, agonists, andantagonists. The term binding site includes a functional binding siteand/or a structural binding site. A structural binding site can include“in contact” amino acid residues as determined from examination of athree-dimensional structure. “Contact” can be determined using Van derWaals radii of atoms or by proximity sufficient to exclude solvent,typically water, from the space between the ligand and the molecule ormolecular complex. In some embodiments, a FGFR3 residue in contact withan anti-FGFR3 antibody (e.g., YW184.6) or other substrate or inhibitoris a residue that has one atom within about 5 Å of an anti-FGFR3antibody residue. Alternatively, “in contact” residue may be those thathave a loss of solvent accessible surface area of at least about 10 Åand, more preferably at least about 50 Å to about 300 Å. Loss of solventaccessible surface can be determined by the method of Lee & Richards (JMol Biol. 1971 Feb. 14; 55(3):379-400) and similar algorithms known tothose skilled in the art, for instance as found in the SOLV module fromC. Broger of F. Hoffman-La Roche in Basel Switzerland.

Some of the “in contact” amino acid residues, if substituted withanother amino acid type, may not cause any change in a biochemicalassay, a cell-based assay, or an in vivo assay used to define afunctional binding site but may contribute to the formation of a threedimensional structure. A functional binding site includes amino acidresidues that are identified as binding site residues based upon loss orgain of function, for example, loss of binding to ligand upon mutationof the residue. In some embodiments, the amino acid residues of afunctional binding site are a subset of the amino acid residues of thestructural binding site.

The term “FGFR3 binding site” refers to a region of FGFR3 that canfavorably associate with a ligand. In some embodiments, the FGFR3binding site may comprise, consist essentially of, or consist of one ormore of the amino acid residues 154, 155, 158, 159, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 177, 202, 205,207, 210, 212, 214, 216, 217, 241, 246, 247, 248, 278, 279, 280, 281,282, 283, 314, 315, 316, 317 and/or 318 of FGFR3-IIIb polypeptide (e.g.,human FGFR3-IIIb disclosed in UniProKB/Swiss-Prot accession numberP22607_(—)2), and mixtures thereof, or equivalent residues of FGFR3-IIIcpolypeptide (e.g., human FGFR3-IIIc disclosed in UniProKB/Swiss-Protaccession number P22607). In some embodiments, the FGFR3 binding sitemay comprise, consist essentially of, or consist of one or more of theamino acid residues 158, 159, 169, 170, 171, 173, 175, 205, 207, and/or315 of FGFR3-IIIb polypeptide, and mixtures thereof, or equivalentresidues of FGFR3-IIIc polypeptide. In some embodiments, the FGFR3binding site may comprise, consist essentially of, or consist of one ormore of the amino acid residues 158, 170, 171, 173, 175, and/or 315, ormixtures thereof, of FGFR3-IIIb polypeptide, or equivalent residues ofFGFR3-IIIc polypeptide. A structurally equivalent ligand binding site isdefined by a root mean square deviation from the structure coordinatesof the backbone atoms of the amino acids that make up binding sites inFGFR3 for anti-FGFR3 antibody YW184.6 of at most about 0.70 Å,preferably about 5 Å.

“Crystal” as used herein, refers to one form of a solid state of matterin which atoms are arranged in a pattern that repeats periodically inthree-dimensions, typically forming a lattice.

“Complementary or complement” as used herein, means the fit orrelationship between two molecules that permits interaction, includingfor example, space, charge, three-dimensional configuration, and thelike.

The term “corresponding” or “corresponds” refers to an amino acidresidue or amino acid sequence that is found at the same position orpositions in a sequence when the amino acid position or sequences arealigned with a reference sequence. In some embodiments, the referencesequence is a human FGFR3-IIIb disclosed in UniProKB/Swiss-Protaccession number P22607_(—)2) or a human FGFR3-IIIc disclosed inUniProKB/Swiss-Prot accession number P22607. It will be appreciated thatwhen the amino acid position or sequence is aligned with the referencesequence the numbering of the amino acids may differ from that of thereference sequence.

“Heavy atom derivative”, as used herein, means a derivative produced bychemically modifying a crystal with a heavy atom such as Hg, Au, or ahalogen.

“Structural homolog” of FGFR3 as used herein refers to a protein thatcontains one or more amino acid substitutions, deletions, additions, orrearrangements with respect to the amino acid sequence of FGFR3, butthat, when folded into its native conformation, exhibits or isreasonably expected to exhibit at least a portion of the tertiary(three-dimensional) structure of the FGFR3. In some embodiments, aportion of the three dimensional structure refers to structural domainsof the FGFR3, including the an extracellular ligand binding region, withtwo or three immunoglobulin-like domains (IgD1-3), a single-passtransmembrane region, and a cytoplasmic, split tyrosine kinase domain.Homolog tertiary structure can be probed, measured, or confirmed byknown analytic or diagnostic methods, for example, X-ray, NMR, circulardichroism, a panel of monoclonal antibodies that recognize native FGFR3,and like techniques. For example, structurally homologous molecules canhave substitutions, deletions or additions of one or more contiguous ornoncontiguous amino acids, such as a loop or a domain. Structurallyhomologous molecules also include “modified” FGFR3 molecules that havebeen chemically or enzymatically derivatized at one or more constituentamino acid, including side chain modifications, backbone modifications,and N- and C-terminal modifications including acetylation,hydroxylation, methylation, amidation, and the attachment ofcarbohydrate or lipid moieties, cofactors, and like modifications.

“Molecular complex”, as used herein, refers to a combination of boundsubstrate or ligand with polypeptide, such as an antibody bound toFGFR3, or a ligand bound to FGFR3.

“Machine-readable data storage medium”, as used herein, means a datastorage material encoded with machine-readable data, wherein a machineprogrammed with instructions for using such data and is capable ofdisplaying data in the desired format, for example, a graphicalthree-dimensional representation of molecules or molecular complexes.

“Scalable,” as used herein, means the increasing or decreasing ofdistances between coordinates (configuration of points) by a scalarfactor while keeping the angles essentially the same.

“Space group symmetry”, as used herein, means the whole symmetry of thecrystal that combines the translational symmetry of a crystallinelattice with the point group symmetry. A space group is designated by acapital letter identifying the lattice type (P, A, F, etc.) followed bythe point group symbol in which the rotation and reflection elements areextended to include screw axes and glide planes. Note that the pointgroup symmetry for a given space group can be determined by removing thecell centering symbol of the space group and replacing all screw axes bysimilar rotation axes and replacing all glide planes with mirror planes.The point group symmetry for a space group describes the true symmetryof its reciprocal lattice.

“Unit cell”, as used herein, means the atoms in a crystal that arearranged in a regular repeating pattern, in which the smallest repeatingunit is called the unit cell. The entire structure can be reconstructedfrom knowledge of the unit cell, which is characterized by three lengths(a, b and c) and three angles (α, β and γ). The quantities a and b arethe lengths of the sides of the base of the cell and γ is the anglebetween these two sides. The quantity c is the height of the unit cell.The angles α and β describe the angles between the base and the verticalsides of the unit cell.

“X-ray diffraction pattern” means the pattern obtained from X-rayscattering of the periodic assembly of molecules or atoms in a crystal.X-ray crystallography is a technique that exploits the fact that X-raysare diffracted by crystals. X-rays have the proper wavelength (in theÅngström (Å) range, approximately 10-8 cm) to be scattered by theelectron cloud of an atom of comparable size. Based on the diffractionpattern obtained from X-ray scattering of the periodic assembly ofmolecules or atoms in the crystal, the electron density can bereconstructed. Additional phase information can be extracted either fromthe diffraction data or from supplementing diffraction experiments tocomplete the reconstruction (the phase problem in crystallography). Amodel is then progressively built into the experimental electrondensity, refined against the data to produce an accurate molecularstructure.

X-ray structure coordinates define a unique configuration of points inspace. Those of skill in the art understand that a set of structurecoordinates for a protein or a protein/ligand complex, or a portionthereof, define a relative set of points that, in turn, define aconfiguration in three dimensions. A similar or identical configurationcan be defined by an entirely different set of coordinates, provided thedistances and angles between coordinates remain essentially the same. Inaddition, a configuration of points can be defined by increasing ordecreasing the distances between coordinates by a scalar factor, whilekeeping the angles essentially the same.

“Crystal structure” generally refers to the three-dimensional or latticespacing arrangement of repeating atomic or molecular units in acrystalline material. The crystal structure of a crystalline materialcan be determined by X-ray crystallographic methods, see for example,“Principles of Protein X-Ray Crystallography,” by Jan Drenth, SpringerAdvanced Texts in Chemistry, Springer Verlag; 2nd ed., February 1999,ISBN: 0387985875, and “Introduction to Macromolecular Crystallography,”by Alexander McPherson, Wiley-Liss, Oct. 18, 2002, ISBN: 0471251224.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat,” and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain may include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g. residues 82a, 82b, and 82c, etc according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues may be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence.

The phrase “substantially similar,” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (generally one associated with an antibody of theinvention and the other associated with a reference/comparator antibody)such that one of skill in the art would consider the difference betweenthe two values to be of little or no biological and/or statisticalsignificance within the context of the biological characteristicmeasured by said values (e.g., Kd values). The difference between saidtwo values is preferably less than about 50%, preferably less than about40%, preferably less than about 30%, preferably less than about 20%,preferably less than about 10% as a function of the value for thereference/comparator antibody.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Desirably the Kd is 1×10⁻⁷, 1×10⁻⁸, 5×10⁻⁸,1×10⁻⁹, 3×10⁻⁹, 5×10⁻⁹, or even 1×10⁻¹⁰ or stronger. Affinity can bemeasured by common methods known in the art, including those describedherein. Low-affinity antibodies generally bind antigen slowly and tendto dissociate readily, whereas high-affinity antibodies generally bindantigen faster and tend to remain bound longer. A variety of methods ofmeasuring binding affinity are known in the art, any of which can beused for purposes of the present invention. Specific illustrativeembodiments are described in the following.

In one embodiment, the “Kd” or “Kd value” according to this invention ismeasured by a radiolabeled antigen binding assay (RIA) performed withthe Fab version of an antibody of interest and its antigen as describedby the following assay that measures solution binding affinity of Fabsfor antigen by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (Chen, et al., (1999) J. Mol. Biol. 293:865-881).To establish conditions for the assay, microtiter plates (Dynex) arecoated overnight with 5 μg/ml of a capturing anti-Fab antibody (CappelLabs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with2% (w/v) bovine serum albumin in PBS for two to five hours at roomtemperature (approximately 23° C.). In a non-adsorbant plate (Nunc#269620), 100 pM or 26 pM [¹²⁵I]-antigen are mixed with serial dilutionsof a Fab of interest (e.g., consistent with assessment of an anti-VEGFantibody, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599).The Fab of interest is then incubated overnight; however, the incubationmay continue for a longer period (e.g., 65 hours) to insure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1%Tween-20 in PBS. When the plates have dried, 150 μl/well of scintillant(MicroScint-20; Packard) is added, and the plates are counted on aTopcount gamma counter (Packard) for ten minutes. Concentrations of eachFab that give less than or equal to 20% of maximal binding are chosenfor use in competitive binding assays. According to another embodimentthe Kd or Kd value is measured by using surface plasmon resonance assaysusing a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway,N.J.) at 25° C. with immobilized antigen CM5 chips at ˜10 response units(RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcoreIIIc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, into 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at25° C. at a flow rate of approximately 25 μl/min. In some embodiments,the following modifications are used for the surface Plasmon resonanceassay method: antibody is immobilized to CM5 biosensor chips to achieveapproximately 400 RU, and for kinetic measurements, two-fold serialdilutions of target protein (e.g., FGFR3-IIIb or -IIIc) (starting from67 nM) are injected in PBST buffer at 25° C. with a flow rate of about30 ul/minute. Association rates (k_(on)) and dissociation rates(k_(off)) are calculated using a simple one-to-one Langmuir bindingmodel (BIAcore Evaluation Software version 3.2) by simultaneous fittingthe association and dissociation sensorgram. The equilibriumdissociation constant (Kd) is calculated as the ratio k_(off)/k_(on).See, e.g., Chen, Y., et al., (1999) J. Mol. Biol. 293:865-881. If theon-rate exceeds 10⁶ M⁻¹ S⁻¹ by the surface plasmon resonance assayabove, then the on-rate can be determined by using a fluorescentquenching technique that measures the increase or decrease influorescence emission intensity (excitation=295 nm; emission=340 nm, 16nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form) inPBS, pH 7.2, in the presence of increasing concentrations of antigen asmeasured in a spectrometer, such as a stop-flow equipped spectrophometer(Aviv Instruments) or a 8000-series SLM-Aminco spectrophotometer(ThermoSpectronic) with a stir red cuvette.

An “on-rate” or “rate of association” or “association rate” or “k_(on)”according to this invention can also be determined with the same surfaceplasmon resonance technique described above using a BIAcore™-2000 or aBIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. withimmobilized antigen CM5 chips at ˜10 response units (RU). Briefly,carboxymethylated dextran biosensor chips (CM5, BIAcore IIIc.) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, into 5 μg/ml (˜0.2 uM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1M ethanolamine is injectedto block unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%Tween 20 (PBST) at 25° C. at a flow rate of approximately 25 μl/min. Insome embodiments, the following modifications are used for the surfacePlasmon resonance assay method: antibody is immobilized to CM5 biosensorchips to achieve approximately 400 RU, and for kinetic measurements,two-fold serial dilutions of target protein (e.g., FGFR3-IIIb or -IIIc)(starting from 67 nM) are injected in PBST buffer at 25° C. with a flowrate of about 30 ul/minute. Association rates (k_(on)) and dissociationrates (k_(off)) are calculated using a simple one-to-one Langmuirbinding model (BIAcore Evaluation Software version 3.2) by simultaneousfitting the association and dissociation sensorgram. The equilibriumdissociation constant (Kd) was calculated as the ratio k_(off)/k_(on).See, e.g., Chen, Y., et al., (1999) J. Mol. Biol. 293:865-881. However,if the on-rate exceeds 10⁶ M⁻¹ S⁻¹ by the surface plasmon resonanceassay above, then the on-rate is preferably determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow equippedspectrophometer (Aviv Instruments) or a 8000-series SLM-Amincospectrophotometer (ThermoSpectronic) with a stir red cuvette.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase, or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter synthesis, such as by conjugation with a label. Other types ofmodifications include, for example, “caps,” substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotide(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3′ terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars suchas arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs and a basic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), (O)NR₂ (“amidate”), P(O)R,P(O)OR′, CO or CH₂ (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

“Percent (%) amino acid sequence identity” with respect to a peptide orpolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the specific peptide or polypeptide sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. For purposes herein, however, % amino acidsequence identity values are generated using the sequence comparisoncomputer program ALIGN-2, wherein the complete source code for theALIGN-2 program is provided in Table A below. The ALIGN-2 sequencecomparison computer program was authored by Genentech, Inc. and thesource code has been filed with user documentation in the U.S. CopyrightOffice, Washington D.C., 20559, where it is registered under U.S.Copyright Registration No. TXU510087. The ALIGN-2 program is publiclyavailable through Genentech, Inc., South San Francisco, Calif. or may becompiled from the source code provided in, e.g., WO2007/001851. TheALIGN-2 program should be compiled for use on a UNIX operating system,preferably digital UNIX V4.0D. All sequence comparison parameters areset by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A.

In some embodiments, two or more amino acid sequences are at least 50%,60%, 70%, 80%, or 90% identical. In some embodiments, two or more aminoacid sequences are at least 95%, 97%, 98%, 99%, or even 100% identical.Unless specifically stated otherwise, all % amino acid sequence identityvalues used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program.

The term “FGFR3,” as used herein, refers, unless specifically orcontextually indicated otherwise, to any native or variant (whethernative or synthetic) FGFR3 polypeptide (e.g., FGFR3-IIIb isoform orFGFR3-IIIc isoform). The term “native sequence” specifically encompassesnaturally occurring truncated forms (e.g., an extracellular domainsequence or a transmembrane subunit sequence), naturally occurringvariant forms (e.g., alternatively spliced forms) andnaturally-occurring allelic variants. The term “wild-type FGFR3”generally refers to a polypeptide comprising an amino acid sequence of anaturally occurring FGFR3 protein. The term “wild type FGFR3 sequence”generally refers to an amino acid sequence found in a naturallyoccurring FGFR3.

“Ligand”, as used herein, refers to an agent or compound that associateswith a binding site on a molecule, for example, FGFR3 binding sites, andmay be an antagonist or agonist of FGFR3 activity. Ligands includemolecules that mimic anti-FGFR3 antibody (e.g., R3Mab) binding to FGFR3.A ligand may be any native or variant (whether native or synthetic)FGFR3 ligand (for example, FGF1, FGF2, FGF4, FGF8, FGF9, FGF17, FGF18,FGF23) polypeptide. The term “native sequence” specifically encompassesnaturally occurring truncated forms (e.g., an extracellular domainsequence or a transmembrane subunit sequence), naturally occurringvariant forms (e.g., alternatively spliced forms) andnaturally-occurring allelic variants. The term “wild-type FGFR3 ligand”generally refers to a polypeptide comprising an amino acid sequence of anaturally occurring FGFR3 ligand protein. The term “wild type FGFR3ligand sequence” generally refers to an amino acid sequence found in anaturally occurring FGFR3 ligand.

“Compound” refers to molecule that associates with FGFR3 or apharmaceutically acceptable salt, ester, amide, prodrug, isomer, ormetabolite, thereof. “Pharmaceutically acceptable salt” refers to aformulation of a compound that does not compromise the biologicalactivity and properties of the compound. Pharmaceutical salts can beobtained by reacting a binding-active compound of the disclosure withinorganic or organic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and thelike.

“FGFR3 activation” refers to activation, or phosphorylation, of theFGFR3 receptor. Generally, FGFR3 activation results in signaltransduction (e.g. that caused by an intracellular kinase domain of aFGFR3 receptor phosphorylating tyrosine residues in FGFR3 or a substratepolypeptide). FGFR3 activation may be mediated by FGFR ligand binding toa FGFR3 receptor of interest. FGFR3 ligand (e.g., such as FGF1 or FGF9)binding to FGFR3 may activate a kinase domain of FGFR3 and therebyresult in phosphorylation of tyrosine residues in the FGFR3 and/orphosphorylation of tyrosine residues in additional substratepolypeptides(s).

The term “constitutive” as used herein, as for example applied toreceptor kinase activity, refers to continuous signaling activity of areceptor that is not dependent on the presence of a ligand or otheractivating molecules. Depending on the nature of the receptor, all ofthe activity may be constitutive or the activity of the receptor may befurther activated by the binding of other molecules (e.g. ligands).Cellular events that lead to activation of receptors are well knownamong those of ordinary skill in the art. For example, activation mayinclude oligomerization, e.g., dimerization, trimerization, etc., intohigher order receptor complexes. Complexes may comprise a single speciesof protein, i.e., a homomeric complex. Alternatively, complexes maycomprise at least two different protein species, i.e., a heteromericcomplex. Complex formation may be caused by, for example, overexpressionof normal or mutant forms of receptor on the surface of a cell. Complexformation may also be caused by a specific mutation or mutations in areceptor.

The term “ligand-independent” as used herein, as for example applied toreceptor signaling activity, refers to signaling activity that is notdependent on the presence of a ligand. A receptor havingligand-independent kinase activity will not necessarily preclude thebinding of ligand to that receptor to produce additional activation ofthe kinase activity.

The term “ligand-dependent” as used herein, as for example applied toreceptor signaling activity, refers to signaling activity that isdependent on the presence of a ligand.

The term “mutation”, as used herein, means a difference in the aminoacid or nucleic acid sequence of a particular protein or nucleic acid(gene, RNA) relative to the wild-type protein or nucleic acid,respectively. A mutated protein or nucleic acid can be expressed from orfound on one allele (heterozygous) or both alleles (homozygous) of agene, and may be somatic or germ line. In the instant invention,mutations are generally somatic. Mutations include sequencerearrangements such as insertions, deletions, and point mutations(including single nucleotide/amino acid polymorphisms).

To “inhibit” is to decrease or reduce an activity, function, and/oramount as compared to a reference.

An agent possesses “agonist activity or function” when an agent mimicsat least one of the functional activities of a polypeptide of interest(e.g., FGFR ligand, such as FGF1 or FGF9).

An “agonist antibody”, as used herein, is an antibody which mimics atleast one of the functional activities of a polypeptide of interest(e.g., FGFR ligand, such as FGF1 or FGF9).

The term “Fc region”, as used herein, generally refers to a dimercomplex comprising the C-terminal polypeptide sequences of animmunoglobulin heavy chain, wherein a C-terminal polypeptide sequence isthat which is obtainable by papain digestion of an intact antibody. TheFc region may comprise native or variant Fc sequences. Although theboundaries of the Fc sequence of an immunoglobulin heavy chain mightvary, the human IgG heavy chain Fc sequence is usually defined tostretch from an amino acid residue at about position Cys226, or fromabout position Pro230, to the carboxyl terminus of the Fc sequence. TheFc sequence of an immunoglobulin generally comprises two constantdomains, a CH2 domain and a CH3 domain, and optionally comprises a CH4domain. The C-terminal lysine (residue 447 according to the EU numberingsystem) of the Fc region may be removed, for example, duringpurification of the antibody or by recombinant engineering of thenucleic acid encoding the antibody. Accordingly, a compositioncomprising an antibody having an Fc region according to this inventioncan comprise an antibody with K447, with all K447 removed, or a mixtureof antibodies with and without the K447 residue.

By “Fc polypeptide” herein is meant one of the polypeptides that make upan Fc region. An Fc polypeptide may be obtained from any suitableimmunoglobulin, such as IgG₁, IgG₂, IgG₃, or IgG₄ subtypes, IgA, IgE,IgD or IgM. In some embodiments, an Fc polypeptide comprises part or allof a wild type hinge sequence (generally at its N terminus). In someembodiments, an Fc polypeptide does not comprise a functional or wildtype hinge sequence.

A “blocking” antibody or an antibody “antagonist” is one which inhibitsor reduces biological activity of the antigen it binds. Preferredblocking antibodies or antagonist antibodies completely inhibit thebiological activity of the antigen.

A “naked antibody” is an antibody that is not conjugated to aheterologous molecule, such as a cytotoxic moiety or radiolabel.

An antibody having a “biological characteristic” of a designatedantibody is one which possesses one or more of the biologicalcharacteristics of that antibody which distinguish it from otherantibodies that bind to the same antigen.

In order to screen for antibodies which bind to an epitope on an antigenbound by an antibody of interest, a routine cross-blocking assay such asthat described in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.

To increase the half-life of the antibodies or polypeptide containingthe amino acid sequences of this invention, one can attach a salvagereceptor binding epitope to the antibody (especially an antibodyfragment), as described, e.g., in U.S. Pat. No. 5,739,277. For example,a nucleic acid molecule encoding the salvage receptor binding epitopecan be linked in frame to a nucleic acid encoding a polypeptide sequenceof this invention so that the fusion protein expressed by the engineerednucleic acid molecule comprises the salvage receptor binding epitope anda polypeptide sequence of this invention. As used herein, the term“salvage receptor binding epitope” refers to an epitope of the Fc regionof an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, or IgG₄) that is responsiblefor increasing the in vivo serum half-life of the IgG molecule (e.g.,Ghetie et al., Ann. Rev. Immunol. 18:739-766 (2000), Table 1).Antibodies with substitutions in an Fc region thereof and increasedserum half-lives are also described in WO00/42072, WO 02/060919; Shieldset al., J. Biol. Chem. 276:6591-6604 (2001); Hinton, J. Biol. Chem.279:6213-6216 (2004)). In another embodiment, the serum half-life canalso be increased, for example, by attaching other polypeptidesequences. For example, antibodies or other polypeptides useful in themethods of the invention can be attached to serum albumin or a portionof serum albumin that binds to the FcRn receptor or a serum albuminbinding peptide so that serum albumin binds to the antibody orpolypeptide, e.g., such polypeptide sequences are disclosed inWO01/45746. In one preferred embodiment, the serum albumin peptide to beattached comprises an amino acid sequence of DICLPRWGCLW (SEQ IDNO:183). In another embodiment, the half-life of a Fab is increased bythese methods. See also, Dennis et al. J. Biol. Chem. 277:35035-35043(2002) for serum albumin binding peptide sequences.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule that contains, preferably, at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or more of the entire length of the referencenucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30,40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, or morenucleotides or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160,180, 190, 200 amino acids or more.

The phrase “little to no agonist function” with respect to an antibodyof the invention, as used herein, means the antibody does not elicit abiologically meaningful amount of agonist activity, e.g., uponadministration to a subject. As would be understood in the art, amountof an activity may be determined quantitatively or qualitatively, solong as a comparison between an antibody of the invention and areference counterpart can be done. The activity can be measured ordetected according to any assay or technique known in the art,including, e.g., those described herein. The amount of activity for anantibody of the invention and its reference counterpart can bedetermined in parallel or in separate runs. In some embodiments, abivalent antibody of the invention does not possess substantial agonistfunction.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (e.g., full lengthor intact monoclonal antibodies), polyclonal antibodies, multivalentantibodies, multispecific antibodies (e.g., bispecific antibodies solong as they exhibit the desired biological activity) and may alsoinclude certain antibody fragments (as described in greater detailherein). An antibody can be human, humanized, and/or affinity matured.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The

CDRs in each chain are held together in close proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen-binding site of antibodies (see Kabat et al.,Sequences of Proteins of Immunological Interest, Fifth Edition, NationalInstitute of Health, Bethesda, Md. (1991)). The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv species,one heavy- and one light-chain variable domain can be covalently linkedby a flexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these can be further divided into subclasses(isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known. “Antibody fragments” comprise only aportion of an intact antibody, wherein the portion preferably retains atleast one, preferably most or all, of the functions normally associatedwith that portion when present in an intact antibody. Examples ofantibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments. In oneembodiment, an antibody fragment comprises an antigen binding site ofthe intact antibody and thus retains the ability to bind antigen. Inanother embodiment, an antibody fragment, for example one that comprisesthe Fc region, retains at least one of the biological functions normallyassociated with the Fc region when present in an intact antibody, suchas FcRn binding, antibody half life modulation, ADCC function andcomplement binding. In one embodiment, an antibody fragment is amonovalent antibody that has an in vivo half life substantially similarto an intact antibody. For e.g., such an antibody fragment may compriseon antigen binding arm linked to an Fc sequence capable of conferring invivo stability to the fragment.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six hypervariable regions; three in the VH (H1, H2, H3), andthree in the VL (L1, L2, L3). A number of hypervariable regiondelineations are in use and are encompassed herein. The KabatComplementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk, J.Mol. Biol. 196:901-917 (1987)). The AbM hypervariable regions representa compromise between the Kabat CDRs and Chothia structural loops, andare used by Oxford Molecular's AbM antibody modeling software. The“contact” hypervariable regions are based on an analysis of theavailable complex crystal structures. The residues from each of thesehypervariable regions are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101Hypervariable regions may comprise “extended hypervariable regions” asfollows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) in theVL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3)in the VH. The variable domain residues are numbered according to Kabatet al., supra for each of these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994).

“Chimeric” antibodies (immunoglobulins) have a portion of the heavyand/or light chain identical with or homologous to correspondingsequences in antibodies derived from a particular species or belongingto a particular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).Humanized antibody as used herein is a subset of chimeric antibodies.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFvsee Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

An “antigen” is a predetermined antigen to which an antibody canselectively bind. The target antigen may be polypeptide, carbohydrate,nucleic acid, lipid, hapten or other naturally occurring or syntheticcompound. Preferably, the target antigen is a polypeptide.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues.

An “affinity matured” antibody is one with one or more alterations inone or more CDRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). Preferred affinity matured antibodieswill have nanomolar or even picomolar affinities for the target antigen.Affinity matured antibodies are produced by procedures known in the art.Marks et al. Bio/Technology 10:779-783 (1992) describes affinitymaturation by VH and VL domain shuffling. Random mutagenesis of CDRand/or framework residues is described by: Barbas et al., Proc Nat.Acad. Sci, USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155(1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al.,J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol.226:889-896 (1992).

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cellactivation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are absolutely required for such killing. The primary cellsfor mediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cellsis summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, such as that described in U.S. Pat.No. 5,500,362 or 5,821,337 or Presta U.S. Pat. No. 6,737,056 may beperformed. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source, e.g., from blood.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domainInhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see review M. inDaëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed inRavetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. The term also includesthe neonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)) and regulates homeostasis ofimmunoglobulins. WO 00/42072 (Presta) describes antibody variants withimproved or diminished binding to FcRs. The content of that patentpublication is specifically incorporated herein by reference. See, also,Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).

Methods of measuring binding to FcRn are known (see, e.g., Ghetie 1997,Hinton 2004). Binding to human FcRn in vivo and serum half life of humanFcRn high affinity binding polypeptides can be assayed, e.g., intransgenic mice or transfected human cell lines expressing human FcRn,or in primates administered with the Fc variant polypeptides.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996), may be performed.

Polypeptide variants with altered Fc region amino acid sequences andincreased or decreased C1q binding capability are described in U.S. Pat.No. 6,194,551B1 and WO 99/51642. The contents of those patentpublications are specifically incorporated herein by reference. See,also, Idusogie et al., J. Immunol. 164:4178-4184 (2000).

The term “Fc region-comprising polypeptide” refers to a polypeptide,such as an antibody or immunoadhesin, which comprises an Fc region. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during purification of thepolypeptide or by recombinant engineering the nucleic acid encoding thepolypeptide. Accordingly, a composition comprising a polypeptide havingan Fc region according to this invention can comprise polypeptides withK447, with all K447 removed, or a mixture of polypeptides with andwithout the K447 residue.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a VL or VH framework derived froma human immunoglobulin framework, or from a human consensus framework.An acceptor human framework “derived from” a human immunoglobulinframework or human consensus framework may comprise the same amino acidsequence thereof, or may contain pre-existing amino acid sequencechanges. Where pre-existing amino acid changes are present, preferablyno more than 5 and preferably 4 or less, or 3 or less, pre-existingamino acid changes are present. Where pre-existing amino acid changesare present in a VH, preferably those changes are only at three, two, orone of positions 71H, 73H, and 78H; for instance, the amino acidresidues at those positions may be 71A, 73T, and/or 78A. In oneembodiment, the VL acceptor human framework is identical in sequence tothe VL human immunoglobulin framework sequence or human consensusframework sequence.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residue in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al. In one embodiment, for the VL, the subgroup is subgroupkappa I as in Kabat et al. In one embodiment, for the VH, the subgroupis subgroup III as in Kabat et al.

A “VH subgroup III consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable heavy subgroup III ofKabat et al. In one embodiment,

the VH subgroup III consensus framework amino acid sequence comprises atleast a portion or all of each of the following sequences

(SEQ ID NO: 184) EVQLVESGGGLVQPGGSLRLSCAAS-H1- (SEQ ID NO: 185)WVRQAPGKGLEWV-H2- (SEQ ID NO: 186) RFTISRDNSKNTLYLQMNSLRAEDTAVYYC-H3-(SEQ ID NO: 187) WGQGTLVTVSS.

A “VL subgroup I consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable light kappa subgroupI of Kabat et al. In one embodiment, the VH subgroup I consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences:

(SEQ ID NO: 188) DIQMTQSPSSLSASVGDRVTITC-L1- (SEQ ID NO: 189)WYQQKPGKAPKLLIY-L2- (SEQ ID NO: 190)GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC-L3- (SEQ ID NO: 191) FGQGTKVEIK.

As used herein, “antibody mutant” or “antibody variant” refers to anamino acid sequence variant of an antibody wherein one or more of theamino acid residues of the species-dependent antibody have beenmodified. Such mutants necessarily have less than 100% sequence identityor similarity with the species-dependent antibody. In one embodiment,the antibody mutant will have an amino acid sequence having at least 75%amino acid sequence identity or similarity with the amino acid sequenceof either the heavy or light chain variable domain of thespecies-dependent antibody, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, and mostpreferably at least 95%. Identity or similarity with respect to thissequence is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical (i.e. same residue) or similar(i.e. amino acid residue from the same group based on common side-chainproperties, see below) with the species-dependent antibody residues,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity. None of N-terminal,C-terminal, or internal extensions, deletions, or insertions into theantibody sequence outside of the variable domain shall be construed asaffecting sequence identity or similarity

Compositions and Methods of the Invention

The present disclosure includes a crystalline form and a crystalstructure of FGFR3 complexed with an anti-FGFR3 antibody, and methods ofusing the FGFR3:anti-FGFR3 antibody crystal structure and structuralcoordinates to identify homologous proteins and to design or identifyagents that can modulate the function of FGFR3 or the FGFR3-anti-FGFR3antibody complex. The present disclosure also includes thethree-dimensional configuration of points derived from the structurecoordinates of at least a portion of an FGFR3 molecule or molecularcomplex, as well as structurally equivalent configurations, as describedbelow. The three-dimensional configuration includes points derived fromstructure coordinates of, e.g., the FGFR3:anti-FGFR3 antibody complex,representing the locations of a plurality of the amino acids definingthe FGFR3-anti-FGFR3 antibody complex binding site.

In some embodiments, the three-dimensional configuration includes pointsderived from structure coordinates representing the locations of thebackbone atoms of a plurality of amino acids defining theFGFR3-anti-FGFR3 antibody complex or the FGFR3 binding site of, e.g.,the FGFR3:anti-FGFR3 antibody complex. Alternatively, thethree-dimensional configuration includes points derived from structurecoordinates representing the locations of the side chain and thebackbone atoms (other than hydrogens) of a plurality of the amino acidsdefining the FGFR3-anti-FGFR3 antibody complex.

The disclosure also includes the scalable three-dimensionalconfiguration of points derived from structure coordinates of moleculesor molecular complexes that are structurally homologous to FGFR3,anti-FGFR3 antibody, or FGFR3:anti-FGFR3 antibody complex, as well asstructurally equivalent configurations. Structurally homologousmolecules or molecular complexes are defined below. Advantageously,structurally homologous molecules can be identified using the structurecoordinates of the FGFR3, anti-FGFR3 antibody, or FGFR3:anti-FGFR3antibody complex or extracellular fragment(s) of FGFR3 according to amethod of the disclosure.

The configurations of points in space derived from structure coordinatesaccording to the disclosure can be visualized as, for example, aholographic image, a stereodiagram, a model, or a computer-displayedimage, and the disclosure thus includes such images, diagrams or models.

The crystal structure and structural coordinates can be used in methods,for example, for obtaining structural information of a related molecule,and for identifying and designing agents that modulate FGFR3, or theFGFR3:anti-FGFR3 antibody complex.

In some embodiments, the FGFR3 binding site may comprise, consistessentially of, or consist of one or more of the amino acid residues154, 155, 158, 159, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 177, 202, 205, 207, 210, 212, 214, 216, 217,241, 246, 247, 248, 278, 279, 280, 281, 282, 283, 314, 315, 316, 317and/or 318, or mixtures thereof, of FGFR3-IIIb polypeptide (e.g., humanFGFR3-IIIb disclosed in (UniProKB/Swiss-Prot accession numberP22607_(—)2), or equivalent residues of FGFR3-IIIc polypeptide (e.g.,human FGFR3-IIIc disclosed in UniProKB/Swiss-Prot accession numberP22607). In some embodiments, the FGFR3 binding site may comprise,consist essentially of, or consist of one or more of the amino acidresidues 158, 159, 169, 170, 171, 173, 175, 205, 207, and/or 315, ormixtures thereof, of FGFR3-IIIb polypeptide, or equivalent residues ofFGFR3-IIIc polypeptide. In some embodiments, the FGFR3 binding site maycomprise, consist essentially of, or consist of one or more of the aminoacid residues 158, 170, 171, 173, 175, and/or 315, or mixtures thereof,of FGFR3-IIIb polypeptide, or equivalent residues of FGFR3-IIIcpolypeptide.

FGFR3 Polypeptides, Polynucleotides and Variants Thereof

FGFR3 nucleic acid and amino acid sequences are known in the art.Nucleic acid sequence encoding the FGFR3 can be designed using the aminoacid sequence of the desired region of FGFR3. As is well-known in theart, there are two major splice isoforms of FGFR3, FGFR3-IIIb andFGFR3-IIIc. FGFR3 sequences are well-known in the art and may includethe sequence of UniProKB/Swiss-Prot accession number P22607 (FGFR3-IIIc)or P22607_(—)2 (FGFR3-IIIb). FGFR3 mutations have been identified andare well-known in the art and include the following mutations (withreference to the sequences shown in UniProKB/Swiss-Prot accession numberP22607 (FGFR3-IIIc) or P22607_(—)2 (FGFR3-IIIb):

FGFR3-IIIb FGFR3-IIIc R248C R248C S249C S249C G372C G370C Y375C Y373CG382R G380R K652E K650E

The present disclosure also includes a polypeptides comprising,consisting essentially of, or consisting of a portion or fragment of theFGFR3, and polynucleotides encoding the polypeptides.

An embodiment of a polypeptide fragment comprises, consists essentiallyof, or consists of any of amino acid residue starting from amino acidresidue 154 to amino acid residue 164 and ending at amino acid residue178 to amino acid 283 of human FGFR3 (e.g., human UniProKB/Swiss-Protaccession number P22607_(—)2 (human FGFR3-IIIb)). An embodiment of apolypeptide fragment comprises, consists essentially of, or consists ofany of amino acid residue 154 to amino acid residue 177, amino acidresidue 202 to amino acid reside 217, amino acid residue 241 to aminoacid residu3 248, amino acid residue 278 to amino acid residue 283and/or amino acid residue 314 to amino acid residue 318. An embodimentof a polypeptide fragment comprises, consists essentially of, orconsists of any of amino acid residue 164 to amino acid residue 164 toresidue 178, residue 269 to residue 283 and/or residue 154 to residue318. In some embodiments, the polypeptide portion has the ability tobind to FGFR3 ligand.

The present disclosure also includes variants of FGFR3. Variants includethose polypeptides that have amino acid substitutions, deletions, andadditions (such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9 10, or more aminoacid substitutions, deletions and additions). In some embodiments, thevariant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 10 or more conservativesubstitutions (relative to a reference sequence, such as a human FGFR3reference sequence). Amino acid substitutions can be made for example toreplace cysteines and eliminate formation of disulfide bonds. Amino acidsubstitutions can also be made to change proteolytic cleavage sites.Other variants can be made at the FGFR3 inhibitor binding site. In otherembodiments, the variants of the FGFR3 bind FGFR3 ligand with the sameor higher affinity than the wild type FGFR3. In some embodiments, thevariants of the FGFR3 bind an FGFR3 inhibitor (e.g. anti-FGFR3 antibody)with the same or higher affinity that the wild type FGFR3.

Fusion Proteins

FGFR3 polypeptides, variants, or structural homolog or portions thereof,may be fused to a heterologous polypeptide or compound. The heterologouspolypeptide is a polypeptide that has a different function than that ofthe FGFR3. Examples of heterologous polypeptide include polypeptidesthat may act as carriers, may extend half life, may act as epitope tags,may provide ways to detect or purify the fusion protein. Heterologouspolypeptides include KLH, albumin, salvage receptor binding epitopes,immunoglobulin constant regions, and peptide tags. Peptide tags usefulfor detection or purification include FLAG, gD protein, polyhistidinetags, hemagluthinin from influenza virus, T7 tag, S tag, Strep tag,chloramiphenicol acetyl transferase, biotin, glutathione-S transferase,green fluorescent protein and maltose binding protein. Compounds thatcan be combined with the FGFR3, variants or structural homolog orportions thereof, include radioactive labels, protecting groups, andcarbohydrate or lipid moieties.

Polynucleotides, Vectors and Host Cells

FGFR3, variants or fragments thereof can be prepared by introducingappropriate nucleotide changes into DNA encoding FGFR3, or by synthesisof the desired polypeptide variants.

Polynucleotide sequences encoding the polypeptides described herein canbe obtained using standard recombinant techniques. Desiredpolynucleotide sequences may be isolated and sequenced from appropriatesource cells. Alternatively, polynucleotides can be synthesized usingnucleotide synthesizer or PCR techniques. Once obtained, sequencesencoding the polypeptides or variant polypeptides are inserted into arecombinant vector capable of replicating and expressing heterologouspolynucleotides in a host cell. Many vectors that are available andknown in the art can be used for the purpose of the present invention.Selection of an appropriate vector will depend mainly on the size of thenucleic acids to be inserted into the vector and the particular hostcell to be transformed with the vector. Each vector contains variouscomponents, depending on its function (amplification or expression ofheterologous polynucleotide, or both) and its compatibility with theparticular host cell in which it resides. The vector componentsgenerally include, but are not limited to: an origin of replication (inparticular when the vector is inserted into a prokaryotic cell), aselection marker gene, a promoter, a ribosome binding site (RBS), asignal sequence, the heterologous nucleic acid insert and atranscription termination sequence.

In general, plasmid vectors containing replicon and control sequences,which are derived from a species compatible with the host cell are usedin connection with these hosts. The vector ordinarily carries areplication site, as well as marking sequences, which are capable ofproviding phenotypic selection in transformed cells. For example, E.coli is typically transformed using pBR322, a plasmid derived from an E.coli species. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM.TM.-11 may be utilized in making arecombinant vector which can be used to transform susceptible host cellssuch as E. coli LE392.

Either constitutive or inducible promoters can be used in the presentinvention, in accordance with the needs of a particular situation, whichcan be ascertained by one skilled in the art. A large number ofpromoters recognized by a variety of potential host cells are wellknown. The selected promoter can be operably linked to cistron DNAencoding a polypeptide described herein by removing the promoter fromthe source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector of choice. Both the nativepromoter sequence and many heterologous promoters may be used to directamplification and/or expression of the target genes. However,heterologous promoters are preferred, as they generally permit greatertranscription and higher yields of expressed target gene as compared tothe native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the β-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to cistrons encoding the polypeptides orvariant polypeptides (Siebenlist et al. (1980) Cell 20: 269) usinglinkers or adaptors to supply any required restriction sites.

In embodiments, each cistron within a recombinant vector comprises asecretion signal sequence component that directs translocation of theexpressed polypeptides across a membrane. In general, the signalsequence may be a component of the vector, or it may be a part of thepolypeptide encoding DNA that is inserted into the vector. The signalsequence selected for the purpose of this invention should be one thatis recognized and processed (i.e. cleaved by a signal peptidase) by thehost cell. For prokaryotic host cells that do not recognize and processthe signal sequences native to the heterologous polypeptides, the signalsequence is substituted by a prokaryotic signal sequence selected, forexample, from the group consisting of the alkaline phosphatase,penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB,PhoE, PelB, OmpA and MBP.

Prokaryotic host cells suitable for expressing polypeptides includeArchaebacteria and Eubacteria, such as Gram-negative or Gram-positiveorganisms. Examples of useful bacteria include Escherichia (e.g., E.coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species(e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans,Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus.Preferably, gram-negative cells are used. Preferably the host cellshould secrete minimal amounts of proteolytic enzymes, and additionalprotease inhibitors may desirably be incorporated in the cell culture.

Besides prokaryotic host cells, eukaryotic host cell systems are alsowell established in the art. Examples of invertebrate cells includeinsect cells such as Drosophila S2 and Spodoptera Sf9, as well as plantsand plant cells. Examples of useful mammalian host cell lines includeChinese hamster ovary (CHO) and COS cells. More specific examplesinclude monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc.Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather,Biol. Reprod., 23:243-251 (1980)); and mouse mammary tumor (MMT 060562,ATCC CCL51).

Polypeptide Production

Host cells are transformed or transfected with the above-describedexpression vectors and cultured in conventional nutrient media modifiedas appropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO4 precipitation and electroporation. Successfultransfection is generally recognized when any indication of theoperation of this vector occurs within the host cell.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include luria broth (LB) plusnecessary nutrient supplements. In preferred embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, the preferred temperature ranges from about20° C. to about 39° C., more preferably from about 25° C. to about 37°C., even more preferably at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH is preferably from about 6.8 to about 7.4,and more preferably about 7.0.

If an inducible promoter is used in the expression vector, proteinexpression is induced under conditions suitable for the activation ofthe promoter. For example, if a PhoA promoter is used for controllingtranscription, the transformed host cells may be cultured in aphosphate-limiting medium for induction. A variety of other inducers maybe used, according to the vector construct employed, as is known in theart.

Eukaryotic host cells are cultured under conditions suitable forexpression of the FGFR3 and/or KD polypeptides. The host cells used toproduce the polypeptides may be cultured in a variety of media.Commercially available media such as Ham's F10 (Sigma), MinimalEssential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco'sModified Eagle's Medium ((DMEM), Sigma) are suitable for culturing thehost cells. In addition, any of the media described in one or more ofHam et al., 1979, Meth. Enz. 58:44, Barnes et al., 1980, Anal. Biochem.102: 255, U.S. Pat. No. 4,767,704, U.S. Pat. No. 4,657,866, U.S. Pat.No. 4,927,762, U.S. Pat. No. 4,560,655, or U.S. Pat. No. 5,122,469, WO90/103430, WO 87/00195, and U.S. Pat. No. Re. 30,985 may be used asculture media for the host cells. Any of these media may be supplementedas necessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES™),nucleotides (such as adenosine and thymidine), antibiotics (such asGENTAMYCIN™), trace elements (defined as inorganic compounds usuallypresent at final concentrations in the micromolar range), and glucose oran equivalent energy source. Other supplements may also be included atappropriate concentrations that would be known to those skilled in theart. The culture conditions, such as temperature, pH, and the like, arethose previously used with the host cell selected for expression, andwill be apparent to the ordinarily skilled artisan.

Polypeptides described herein expressed in a host cell may be secretedinto and/or recovered from the periplasm of the host cells. Proteinrecovery typically involves disrupting the microorganism, generally bysuch means as osmotic shock, sonication or lysis. Once cells aredisrupted, cell debris or whole cells may be removed by centrifugationor filtration. The proteins may be further purified, for example, byaffinity resin chromatography. Alternatively, proteins can betransported into the culture media and isolated there from. Cells may beremoved from the culture and the culture supernatant being filtered andconcentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as fractionation on immunoaffinity orion-exchange columns; ethanol precipitation; reverse phase HPLC;chromatography on silica or on a cation exchange resin such as DEAE;chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75; hydrophobic affinityresins, ligand affinity using a suitable antigen immobilized on a matrixand Western blot assay.

Polypeptides that are produced may be purified to obtain preparationsthat are substantially homogeneous for further assays and uses. Standardprotein purification methods known in the art can be employed. Thefollowing procedures are exemplary of suitable purification procedures:fractionation on immunoaffinity or ion-exchange columns, ethanolprecipitation, reverse phase HPLC, chromatography on silica or on acation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammoniumsulfate precipitation, and gel filtration using, for example, SephadexG-75.

Antibody Production

For recombinant production of an antibody, the nucleic acid encoding itis isolated and inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. DNA encoding the antibodyis readily isolated and sequenced using conventional procedures (e.g.,by using oligonucleotide probes that are capable of binding specificallyto genes encoding the heavy and light chains of the antibody). Manyvectors are available. The choice of vector depends in part on the hostcell to be used. Generally, preferred host cells are of eitherprokaryotic or eukaryotic (generally mammalian) origin.

Generating Antibodies Using Prokaryotic Host Cells:

Vector Construction

Polynucleotide sequences encoding polypeptide components of the antibodyof the invention can be obtained using standard recombinant techniques.Desired polynucleotide sequences may be isolated and sequenced fromantibody producing cells such as hybridoma cells. Alternatively,polynucleotides can be synthesized using nucleotide synthesizer or PCRtechniques. Once obtained, sequences encoding the polypeptides areinserted into a recombinant vector capable of replicating and expressingheterologous polynucleotides in prokaryotic hosts. Many vectors that areavailable and known in the art can be used for the purpose of thepresent invention. Selection of an appropriate vector will depend mainlyon the size of the nucleic acids to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components, depending on its function (amplification orexpression of heterologous polynucleotide, or both) and itscompatibility with the particular host cell in which it resides. Thevector components generally include, but are not limited to: an originof replication, a selection marker gene, a promoter, a ribosome bindingsite (RBS), a signal sequence, the heterologous nucleic acid insert anda transcription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as πGEM.TM.-11 may be utilized in making arecombinant vector which can be used to transform susceptible host cellssuch as E. coli LE392.

The expression vector of the invention may comprise two or morepromoter-cistron pairs, encoding each of the polypeptide components. Apromoter is an untranslated regulatory sequence located upstream (5′) toa cistron that modulates its expression. Prokaryotic promoters typicallyfall into two classes, inducible and constitutive. Inducible promoter isa promoter that initiates increased levels of transcription of thecistron under its control in response to changes in the culturecondition, e.g. the presence or absence of a nutrient or a change intemperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector of the invention. Both thenative promoter sequence and many heterologous promoters may be used todirect amplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the β-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to cistrons encoding the target light andheavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers oradaptors to supply any required restriction sites.

In one aspect of the invention, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected for the purpose of this inventionshould be one that is recognized and processed (i.e. cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the signal sequences native to the heterologouspolypeptides, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group consisting of thealkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II(STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of theinvention, the signal sequences used in both cistrons of the expressionsystem are STII signal sequences or variants thereof.

In another aspect, the production of the immunoglobulins according tothe invention can occur in the cytoplasm of the host cell, and thereforedoes not require the presence of secretion signal sequences within eachcistron. In that regard, immunoglobulin light and heavy chains areexpressed, folded and assembled to form functional immunoglobulinswithin the cytoplasm. Certain host strains (e.g., the E. colitrxB-strains) provide cytoplasm conditions that are favorable fordisulfide bond formation, thereby permitting proper folding and assemblyof expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

The present invention provides an expression system in which thequantitative ratio of expressed polypeptide components can be modulatedin order to maximize the yield of secreted and properly assembledantibodies of the invention. Such modulation is accomplished at least inpart by simultaneously modulating translational strengths for thepolypeptide components.

One technique for modulating translational strength is disclosed inSimmons et al., U.S. Pat. No. 5,840,523. It utilizes variants of thetranslational initiation region (TIR) within a cistron. For a given TIR,a series of amino acid or nucleic acid sequence variants can be createdwith a range of translational strengths, thereby providing a convenientmeans by which to adjust this factor for the desired expression level ofthe specific chain. TIR variants can be generated by conventionalmutagenesis techniques that result in codon changes which can alter theamino acid sequence, although silent changes in the nucleotide sequenceare preferred. Alterations in the TIR can include, for example,alterations in the number or spacing of Shine-Dalgarno sequences, alongwith alterations in the signal sequence. One method for generatingmutant signal sequences is the generation of a “codon bank” at thebeginning of a coding sequence that does not change the amino acidsequence of the signal sequence (i.e., the changes are silent). This canbe accomplished by changing the third nucleotide position of each codon;additionally, some amino acids, such as leucine, serine, and arginine,have multiple first and second positions that can add complexity inmaking the bank. This method of mutagenesis is described in detail inYansura et al. (1992) METHODS: A Companion to Methods in Enzymol.4:151-158.

Preferably, a set of vectors is generated with a range of TIR strengthsfor each cistron therein. This limited set provides a comparison ofexpression levels of each chain as well as the yield of the desiredantibody products under various TIR strength combinations. TIR strengthscan be determined by quantifying the expression level of a reporter geneas described in detail in Simmons et al. U.S. Pat. No. 5,840,523. Basedon the translational strength comparison, the desired individual TIRsare selected to be combined in the expression vector constructs of theinvention.

Prokaryotic host cells suitable for expressing antibodies of theinvention include Archaebacteria and Eubacteria, such as Gram-negativeor Gram-positive organisms. Examples of useful bacteria includeEscherichia (e.g., E. coli), Bacilli (e.g., B. subtilis),Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonellatyphimurium, Serratia marcescans, Klebsiella, Proteus, Shigella,Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negativecells are used. In one embodiment, E. coli cells are used as hosts forthe invention. Examples of E. coli strains include strain W3110(Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.:American Society for Microbiology, 1987), pp. 1190-1219; ATCC DepositNo. 27,325) and derivatives thereof, including strain 33D3 havinggenotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41kanR (U.S. Pat. No. 5,639,635). Other strains and derivatives thereof,such as E. coli 294 (ATCC 31,446), E. coli B, E. coli, 1776 (ATCC31,537) and E. coli RV308 (ATCC 31,608) are also suitable. Theseexamples are illustrative rather than limiting. Methods for constructingderivatives of any of the above-mentioned bacteria having definedgenotypes are known in the art and described in, for example, Bass etal., Proteins, 8:309-314 (1990). It is generally necessary to select theappropriate bacteria taking into consideration replicability of thereplicon in the cells of a bacterium. For example, E. coli, Serratia, orSalmonella species can be suitably used as the host when well knownplasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supplythe replicon. Typically the host cell should secrete minimal amounts ofproteolytic enzymes, and additional protease inhibitors may desirably beincorporated in the cell culture.

Antibody Production

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include luria broth (LB) plusnecessary nutrient supplements. In some embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, the preferred temperature ranges from about20° C. to about 39° C., more preferably from about 25° C. to about 37°C., even more preferably at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH is preferably from about 6.8 to about 7.4,and more preferably about 7.0.

If an inducible promoter is used in the expression vector of theinvention, protein expression is induced under conditions suitable forthe activation of the promoter. In one aspect of the invention, PhoApromoters are used for controlling transcription of the polypeptides.Accordingly, the transformed host cells are cultured in aphosphate-limiting medium for induction. Preferably, thephosphate-limiting medium is the C.R.A.P medium (see, for e.g., Simmonset al., J. Immunol. Methods (2002), 263:133-147). A variety of otherinducers may be used, according to the vector construct employed, as isknown in the art.

In one embodiment, the expressed polypeptides of the present inventionare secreted into and recovered from the periplasm of the host cells.Protein recovery typically involves disrupting the microorganism,generally by such means as osmotic shock, sonication or lysis. Oncecells are disrupted, cell debris or whole cells may be removed bycentrifugation or filtration. The proteins may be further purified, forexample, by affinity resin chromatography. Alternatively, proteins canbe transported into the culture media and isolated therein. Cells may beremoved from the culture and the culture supernatant being filtered andconcentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as polyacrylamide gel electrophoresis (PAGE)and Western blot assay.

In one aspect of the invention, antibody production is conducted inlarge quantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least 1000 liters ofcapacity, preferably about 1,000 to 100,000 liters of capacity. Thesefermentors use agitator impellers to distribute oxygen and nutrients,especially glucose (the preferred carbon/energy source). Small scalefermentation refers generally to fermentation in a fermentor that is nomore than approximately 100 liters in volumetric capacity, and can rangefrom about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD550 of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of theinvention, various fermentation conditions can be modified. For example,to improve the proper assembly and folding of the secreted antibodypolypeptides, additional vectors overexpressing chaperone proteins, suchas Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (apeptidylprolyl cis,trans-isomerase with chaperone activity) can be usedto co-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.(1999) J Bio Chem 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al. (1998),supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S.Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72(1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system of the invention.

Antibody Purification

In one embodiment, the antibody protein produced herein is furtherpurified to obtain preparations that are substantially homogeneous forfurther assays and uses. Standard protein purification methods known inthe art can be employed. The following procedures are exemplary ofsuitable purification procedures: fractionation on immunoaffinity orion-exchange columns, ethanol precipitation, reverse phase HPLC,chromatography on silica or on a cation-exchange resin such as DEAE,chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gelfiltration using, for example, Sephadex G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the full length antibody products of theinvention. Protein A is a 41kD cell wall protein from Staphylococcusaureas which binds with a high affinity to the Fc region of antibodies.Lindmark et al (1983) J. Immunol. Meth. 62:1-13. The solid phase towhich Protein A is immobilized is preferably a column comprising a glassor silica surface, more preferably a controlled pore glass column or asilicic acid column. In some applications, the column has been coatedwith a reagent, such as glycerol, in an attempt to prevent nonspecificadherence of contaminants.

As the first step of purification, the preparation derived from the cellculture as described above is applied onto the Protein A immobilizedsolid phase to allow specific binding of the antibody of interest toProtein A. The solid phase is then washed to remove contaminantsnon-specifically bound to the solid phase. Finally the antibody ofinterest is recovered from the solid phase by elution.

Generating antibodies using eukaryotic host cells:

The vector components generally include, but are not limited to, one ormore of the following:

a signal sequence, an origin of replication, one or more marker genes,an enhancer element, a promoter, and a transcription terminationsequence.

(i) Signal Sequence Component

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

(ii) Origin of Replication

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may typicallybe used only because it contains the early promoter.

(iii) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, where relevant, or (c) supply critical nutrients notavailable from complex media.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II, preferably primate metallothionein genes, adenosine deaminase,ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCCCRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the antibodypolypeptide nucleic acid. Promoter sequences are known for eukaryotes.Virtually alleukaryotic genes have an AT-rich region locatedapproximately 25 to 30 bases upstream from the site where transcriptionis initiated. Another sequence found 70 to 80 bases upstream from thestart of transcription of many genes is a CNCAAT region where N may beany nucleotide. At the 3′ end of most eukaryotic genes is an AATAAAsequence that may be the signal for addition of the poly A tail to the3′ end of the coding sequence. All of these sequences are suitablyinserted into eukaryotic expression vectors.

Antibody polypeptide transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

(v) Enhancer Element Component

Transcription of DNA encoding the antibody polypeptide of this inventionby higher eukaryotes is often increased by inserting an enhancersequence into the vector. Many enhancer sequences are now known frommammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theantibody polypeptide-encoding sequence, but is preferably located at asite 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells will typically alsocontain sequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of eukaryotic or viral DNAsor cDNAs. These regions contain nucleotide segments transcribed aspolyadenylated fragments in the untranslated portion of the mRNAencoding an antibody. One useful transcription termination component isthe bovine growth hormone polyadenylation region. See WO94/11026 and theexpression vector disclosed therein.

(vii) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include higher eukaryote cells described herein, includingvertebrate host cells. Propagation of vertebrate cells in culture(tissue culture) has become a routine procedure. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(viii) Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

(ix) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, or directly secreted into the medium. If the antibodyis produced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, are removed, for example, bycentrifugation or ultrafiltration. Where the antibody is secreted intothe medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX™ resin (J.T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Activity Assays

The antibodies can be characterized for their physical/chemicalproperties and biological functions by various assays known in the art.

The purified immunoglobulins can be further characterized by a series ofassays including, but not limited to, N-terminal sequencing, amino acidanalysis, non-denaturing size exclusion high pressure liquidchromatography (HPLC), mass spectrometry, ion exchange chromatographyand papain digestion.

In certain embodiments of the invention, the immunoglobulins producedherein are analyzed for their biological activity. In some embodiments,the immunoglobulins of the present invention are tested for theirantigen binding activity. The antigen binding assays that are known inthe art and can be used herein include without limitation any direct orcompetitive binding assays using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, fluorescent immunoassays, andprotein A immunoassays.

2. Crystals and Crystal Structures

The present disclosure provides crystals of an FGFR3:anti-FGFR3 antibodycomplex as well as the crystal structure of FGFR3:anti-FGFR3 antibody asdetermined therefrom. In some embodiments, the crystals are formed froman FGFR3 sequence comprising sequence

ADPDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKSWISESVEADVRLRLANVSERDGGEYLCRATNFIGVAEKAFWLSVHGPRAAEEELVEA DEAGSV (SEQID NO:272) and an anti-FGFR3 antibody.

In some embodiments, the crystals are formed from an FGFR3 sequencecomprising sequence

ADPDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKSWISESVEADVRLRLANVSERDGGEYLCRATNFIGVAEKAFWLSVHGPRAAEEELVEA DEAGSVHHHHHH(SEQ ID NO:273) and an anti-FGFR3 antibody.

In some embodiments, the anti-FGFR3 antibody comprises a light chainvariable region comprising HVR-L1, HVR-L2, HVR-L3, wherein each, inorder, comprises SEQ ID NO:4, 5, 6, and/or a heavy chain variable regioncomprising HVR-H1, HVR-H2, and HVR-H3, where each, in order, containsSEQ ID NO: 1, 2, 3. In some embodiments, the anti-FGFR3 antibodycomprises a light chain variable region comprising sequence

(SEQ ID NO: 274) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQ GTKVEIKRand a heavy chain variable region comprising sequence

(SEQ ID NO: 275) EVQLVESGGGLVQPGGSLRLSCAASGFTFTSTGISWVRQAPGKGLEWVGRIY PTN GSTNYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARARTYGIYDLYVDYTEYVMDYWGQGTLV.

In some embodiments, the anti-FGFR3 antibody comprises any antibodydisclosed herein or disclosed in co-pending co-owned U.S. Ser. No.______, filed Mar. 24, 2010 (attorney docket P4294R1).

In a specific embodiment, the structure of FGFR3 complexed with ananti-FGFR3 antibody was solved by molecular replacement with the programPHASER. The crystals belonged to space group P2₁2₁2₁ with cellparameters of a=58.5 Å, b=99.3 Å and c=143.7 Å.

The crystals are useful to provide the crystal structure and to providea stable form of the molecule for storage.

Each of the constituent amino acids of FGFR3:anti-FGFR3 antibody isdefined by a set of structure coordinates as set forth in Table 6. Theterm “structure coordinates” refers to Cartesian coordinates derivedfrom mathematical equations related to the patterns obtained ondiffraction of a monochromatic beam of X-rays by the atoms (scatteringcenters) of a FGFR3 and FGFR3:anti-FGFR3 antibody, in crystal form. Thediffraction data are used to calculate an electron density map of therepeating unit of the crystal. The electron density maps are then usedto establish the positions of the individual atoms of the FGFR3,anti-FGFR3 antibody and FGFR3:anti-FGFR3 antibody complex.

Slight variations in structure coordinates can be generated bymathematically manipulating the FGFR3 and FGFR3:anti-FGFR3 antibodycomplex structure coordinates. For example, the structure coordinates asset forth in Table 6 could be manipulated by crystallographicpermutations of the structure coordinates, fractionalization of thestructure coordinates, integer additions or subtractions to sets of thestructure coordinates, inversion of the structure coordinates, or anycombination of the above. Alternatively, modifications in the crystalstructure due to mutations, additions, substitutions, deletions, andcombinations thereof, of amino acids, or other changes in any of thecomponents that make up the crystal, could also yield variations instructure coordinates. Such slight variations in the individualcoordinates will have little effect on overall shape. If such variationsare within an acceptable standard error as compared to the originalcoordinates, the resulting three-dimensional shape is considered to bestructurally equivalent. Structural equivalence is described in moredetail below.

It should be noted that slight variations in individual structurecoordinates of the FGFR3, anti-FGFR3 antibody and FGFR3:anti-FGFR3antibody complex would not be expected to significantly alter the natureof chemical entities such as ligands that could associate with a bindingsite or other structural features of FGFR3. In this context, the phrase“associating with” refers to a condition of proximity between a ligand,or portions thereof, and a FGFR3 molecule or portions thereof. Theassociation may be non-covalent, wherein the juxtaposition isenergetically favored by hydrogen bonding, van der Waals forces, and/orelectrostatic interactions, or it may be covalent.

FGFR3 residues that form a binding site for a modulator (e.g., anantagonist or agonist) of FGFR3 are described in the presentapplication. The identification of a binding site for a modulator onFGFR3 can be used to design new classes of FGFR3 modulators, such asantagonists, agonists, and like agents, having therapeutic applications,such as, for treating cancer.

3. Structurally Equivalent Crystal Structures

Various computational analyses can be used to determine whether amolecule or portions of the molecule defining structure features are“structurally equivalent,” defined in terms of its three-dimensionalstructure, to all or part of an activated unbound FGFR3 or FGFR3 boundto an inhibitor, such as an anti-FGFR3 antibody, or FGFR3 present inFGFR3:anti-FGFR3 antibody complex. Such analyses may be carried out incurrent software applications, such as the Molecular Similarityapplication of QUANTA (Molecular Simulations IIIc., San Diego, Calif.),Version 4.1, and as described in the accompanying User's Guide.

The Molecular Similarity application permits comparisons betweendifferent structures, different conformations of the same structure, anddifferent parts of the same structure. A procedure used in MolecularSimilarity to compare structures comprises: 1) loading the structures tobe compared; 2) defining the atom equivalences in these structures; 3)performing a fitting operation; and 4) analyzing the results.

One structure is identified as the target (i.e., the fixed structure);all remaining structures are working structures (i.e., movingstructures). Since atom equivalency within QUANTA is defined by userinput, for the purpose of this disclosure equivalent atoms are definedas protein backbone atoms (N, Cα, C, and O) for all conserved residuesbetween the two structures being compared. A conserved residue isdefined as a residue that is structurally or functionally equivalent.Only rigid fitting operations are considered.

When a rigid fitting method is used, the working structure is translatedand rotated to obtain an optimum fit with the target structure. Thefitting operation uses an algorithm that computes the optimumtranslation and rotation to be applied to the moving structure, suchthat the root mean square difference of the fit over the specified pairsof equivalent atom is an absolute minimum. This number, given inAngstroms, is reported by QUANTA.

Structurally equivalent crystal structures have portions of the twomolecules that are substantially identical, within an acceptable marginof error. The margin of error can be calculated by methods known tothose of skill in the art. In some embodiments, any molecule ormolecular complex or any portion thereof, that has a root mean squaredeviation of conserved residue backbone atoms (N, Cα, C, O) of less thanabout 0.70 Å, preferably 0.5 Å. For example, structurally equivalentmolecules or molecular complexes are those that are defined by theentire set of structure coordinates listed in Table 7 or 8±a root meansquare deviation from the conserved backbone atoms of those amino acidsof not more than 0.70 Å, preferably 0.5 Å. The term “root mean squaredeviation” means the square root of the arithmetic mean of the squaresof the deviations. It is a way to express the deviation or variationfrom a trend or object. For purposes of this disclosure, the “root meansquare deviation” defines the variation in the backbone of a proteinfrom the backbone of FGFR3 complex (as defined by the structurecoordinates of the complex as described herein) or a defining structuralfeature thereof

4. Structurally Homologous Molecules, Molecular Complexes, and CrystalStructures

Structure coordinates can be used to aid in obtaining structuralinformation about another crystallized molecule or molecular complex.The method of the disclosure allows determination of at least a portionof the three-dimensional structure of molecules or molecular complexesthat contain one or more structural features that are similar tostructural features of at least a portion of the FGFR3, anti-FGFR3antibody, or FGFR3:anti-FGFR3 antibody complex. These molecules arereferred to herein as “structurally homologous” to FGFR3, anti-FGFR3antibody, or FGFR3:anti-FGFR3 antibody complex. Similar structuralfeatures can include, for example, regions of amino acid identity,conserved active site or binding site motifs, and similarly arrangedsecondary structural elements (for example, binding sites for FGFR3ligand on FGFR3).

Optionally, structural homology is determined by aligning the residuesof the two amino acid sequences to optimize the number of identicalamino acids along the lengths of their sequences; gaps in either or bothsequences are permitted in making the alignment in order to optimize thenumber of identical amino acids, although the amino acids in eachsequence must nonetheless remain in their proper order. Two amino acidsequences are compared using the BLAST program, version 2.0.9, of theBLAST 2 search algorithm, as described by Tatusova et al., and availableat http:www.ncbi.nlm.nih.gov/BLAST/. Preferably, the default values forall BLAST 2 search parameters are used, including matrix=BLOSUM62; opengap penalty=11, extension gap penalty=1, gap x_dropoff=50, expect=10,wordsize=3, and filter on. In the comparison of two amino acid sequencesusing the BLAST search algorithm, structural similarity is referred toas “identity.” In some embodiments, a structurally homologous moleculeis a protein that has an amino acid sequence having at least 80%identity with a wild type or recombinant amino acid sequence of FGFR3,in some embodiments human FGFR3-IIIb or human FGFR3-IIIc. Morepreferably, a protein that is structurally homologous to FGFR3 includesat least one contiguous stretch of at least 50 amino acids that has atleast 80% amino acid sequence identity with the analogous portion of thewild type or recombinant FGFR3. Methods for generating structuralinformation about the structurally homologous molecule or molecularcomplex are well known and include, for example, molecular replacementtechniques.

Therefore, in another embodiment this disclosure provides a method ofutilizing molecular replacement to obtain structural information about amolecule or molecular complex whose structure is unknown comprising:

(a) generating an X-ray diffraction pattern from a crystallized moleculeor molecular complex of unknown or incompletely known structure; and

(b) applying at least a portion of the structural coordinates of FGFR3complex to the X-ray diffraction pattern to generate a three-dimensionalelectron density map of the molecule or molecular complex whosestructure is unknown or incompletely known.

By using molecular replacement, all or part of the structure coordinatesof FGFR3, anti-FGFR3 antibody, or FGFR3:anti-FGFR3 antibody complex asprovided by this disclosure can be used to determine the unsolvedstructure of a crystallized molecule or molecular complex more quicklyand efficiently than attempting to determine such information ab initio.Coordinates of structural features of FGFR3 can be utilized includingthat of trypsin-like serine protease domain.

Molecular replacement can provide an accurate estimation of the phasesfor an unknown or incompletely known structure. Phases are one factor inequations that are used to solve crystal structures, and this factorcannot be determined directly. Obtaining accurate values for the phases,by methods other than molecular replacement, can be a time-consumingprocess that involves iterative cycles of approximations and refinementsand greatly hinders the solution of crystal structures. However, whenthe crystal structure of a protein containing at least a structurallyhomologous portion has been solved, molecular replacement using theknown structure provide a useful estimate of the phases for the unknownor incompletely known structure.

Thus, this method involves generating a preliminary model of a moleculeor molecular complex whose structure coordinates are unknown, byorienting and positioning the relevant portion of the FGFR3, anti-FGFR3antibody, or FGFR3:anti-FGFR3 antibody complex within the unit cell ofthe crystal of the unknown molecule or molecular complex. Thisorientation or positioning is conducted so as best to account for theobserved X-ray diffraction pattern of the crystal of the molecule ormolecular complex whose structure is unknown. Phases can then becalculated from this model and combined with the observed X-raydiffraction pattern amplitudes to generate an electron density map ofthe structure. This map, in turn, can be subjected to established andwell-known model building and structure refinement techniques to providea final, accurate structure of the unknown crystallized molecule ormolecular complex (see for example, Lattman, 1985. Methods in Enzymology115:55-77).

Structural information about a portion of any crystallized molecule ormolecular complex that is sufficiently structurally homologous to aportion of FGFR3, anti-FGFR3 antibody, or FGFR3:anti-FGFR3 antibodycomplex can be solved by this method. In addition to a molecule thatshares one or more structural features with the FGFR3, such as theextracellular ligand binding region, with two or threeimmunoglobulin-like domains (IgD1-3) and an acid box, and/or FGFR3,anti-FGFR3 antibody, or FGFR3:anti-FGFR3 antibody complex as describedabove, a molecule that has similar bioactivity, such as the same ligandbinding activity as FGFR3 and/or anti-FGFR3 antibody, may also besufficiently structurally homologous to a portion of the FGFR3 and/orantiFGFR3 antibody to permit use of the structure coordinates ofFGFR3:anti-FGFR3 antibody to solve its crystal structure or identifystructural features that are similar to those identified in the FGFR3described herein. It will be appreciated that amino acid residues in thestructurally homologous molecule identified as corresponding to theFGFR3 structural feature may have different amino acid numbering.

In one embodiment of the disclosure, the method of molecular replacementis utilized to obtain structural information about a molecule ormolecular complex, wherein the molecule or molecular complex includes atleast one FGFR3, anti-FGFR3 antibody, or FGFR3:anti-FGFR3 antibodycomplex subunit or homolog. In the context of the present disclosure, a“structural homolog” of the FGFR3, anti-FGFR3 antibody, orFGFR3:anti-FGFR3 antibody complex is a protein that contains one or moreamino acid substitutions, deletions, additions, or rearrangements withrespect to the amino acid sequence of FGFR3, anti-FGFR3 antibody, orFGFR3:anti-FGFR3 antibody complex but that, when folded into its nativeconformation, exhibits or is reasonably expected to exhibit at least aportion of the tertiary (three-dimensional) structure of at least aportion of FGFR3, anti-FGFR3 antibody, or FGFR3:anti-FGFR3 antibodycomplex. A portion of the FGFR3 includes the binding site for an FGFR3inhibitor.

A heavy atom derivative of FGFR3 is also included as a FGFR3 homolog.The term “heavy atom derivative” refers to derivatives of FGFR3,anti-FGFR3 antibody, or FGFR3:anti-FGFR3 antibody complex produced bychemically modifying a crystal of FGFR3 or both. In practice, a crystalis soaked in a solution containing heavy metal atom salts, ororganometallic compounds, e.g., lead chloride, gold thiomalate,thiomersal or uranyl acetate, which can diffuse through the crystal andbind to the surface of the protein. The location(s) of the bound heavymetal atom(s) can be determined by X-ray diffraction analysis of thesoaked crystal. This information, in turn, is used to generate the phaseinformation used to construct three-dimensional structure of the protein(Blundell, et al., 1976, Protein Crystallography, Academic Press, SanDiego, Calif.).

Variants may be prepared, for example, by expression of FGFR3 cDNApreviously altered in its coding sequence by oligonucleotide-directedmutagenesis as described herein. Variants may also be generated bysite-specific incorporation of unnatural amino acids into FGFR3 proteinsusing known biosynthetic methods (Noren, et al., 1989, Science244:182-88). In this method, the codon encoding the amino acid ofinterest in wild-type FGFR3 is replaced by a “blank” nonsense codon,TAG, using oligonucleotide-directed mutagenesis. A suppressor tRNAdirected against this codon is then chemically aminoacylated in vitrowith the desired unnatural amino acid. The aminoacylated tRNA is thenadded to an in vitro translation system to yield a mutant FGFR3 with thesite-specific incorporated unnatural amino acid.

For example, structurally homologous molecules can contain deletions oradditions of one or more contiguous or noncontiguous amino acids, suchas a loop or a domain. Structurally homologous molecules also include“modified” FGFR3, anti-FGFR3 antibody, or FGFR3:anti-FGFR3 antibodycomplex that have been chemically or enzymatically derivatized at one ormore constituent amino acid, including side chain modifications,backbone modifications, and N- and C-terminal modifications includingacetylation, hydroxylation, methylation, amidation, and the attachmentof carbohydrate or lipid moieties, cofactors, and like modifications. Itwill be appreciated that amino acid residues in the structurallyhomologous molecule identified as corresponding to activated FGFR3 orother structural feature of the FGFR3 may have different amino acidnumbering.

The structure coordinates of FGFR3 are also particularly useful to solveor model the structure of crystals of FGFR3, anti-FGFR3 antibody, orFGFR3:anti-FGFR3 antibody complex homologs, which are co-complexed witha variety of ligands (e.g., a ligand binding the antagonist bindingsite). This approach enables the determination of the optimal sites forinteraction between ligand, including candidate FGFR3 ligands. Potentialsites for modification within the various binding sites (such as anFGFR3 binding site) of the molecule can also be identified. Thisinformation provides an additional tool for determining more efficientbinding interactions, for example, increased hydrophobic or polarinteractions, between FGFR3 and a ligand. For example, high-resolutionX-ray diffraction data collected from crystals exposed to differenttypes of solvent allows the determination of where each type of solventmolecule resides. Small molecules that bind tightly to those sites canthen be designed and synthesized and tested for their FGFR3 affinity,and/or inhibition activity.

All of the complexes referred to above may be studied using well-knownX-ray diffraction techniques and may be refined versus 1.5-3.5 Åresolution X-ray data to an R-factor of about 0.30 or less usingcomputer software, such as X-PLOR (Yale University, distributed byMolecular Simulations, Inc.) (see for example, Blundell, et al. 1976.Protein Crystallography, Academic Press, San Diego, Calif., and Methodsin Enzymology, Vol. 114 & 115, H. W. Wyckoff et al., eds., AcademicPress (1985)). This information may thus be used to optimize known FGFR3modulators, and more importantly, to design new FGFR3 modulators.

The disclosure also includes the unique three-dimensional configurationdefined by a set of points defined by the structure coordinates for amolecule or molecular complex structurally homologous to FGFR3,anti-FGFR3 antibody, or FGFR3:anti-FGFR3 antibody complex as determinedusing the method of the present disclosure, structurally equivalentconfigurations, and magnetic storage media including such set ofstructure coordinates.

5. Homology Modeling

Using homology modeling, a computer model of a homolog, e.g., an FGFR3homolog, can be built or refined without crystallizing the homolog.First, a preliminary model of the homolog is created by sequencealignment with FGFR3, anti-FGFR3 antibody, or FGFR3:anti-FGFR3 antibodycomplex secondary structure prediction, the screening of structurallibraries, or any combination of those techniques. Computationalsoftware may be used to carry out the sequence alignments and thesecondary structure predictions. Structural incoherences, e.g.,structural fragments around insertions and deletions, can be modeled byscreening a structural library for peptides of the desired length andwith a suitable conformation. For prediction of the side chainconformation, a side chain rotamer library may be employed. If thehomolog has been crystallized, the final homology model can be used tosolve the crystal structure of the homolog by molecular replacement, asdescribed above. Next, the preliminary model is subjected to energyminimization to yield an energy-minimized model. The energy-minimizedmodel may contain regions where stereochemistry restraints are violated,in which case such regions are remodeled to obtain a final homologymodel. The homology model is positioned according to the results ofmolecular replacement, and subjected to further refinement includingmolecular dynamics calculations.

6. Methods for Identification of Modulators of FGFR3

Potent and selective ligands that modulate activity (antagonists andagonists) can be identified using the three-dimensional model of theFGFR3 using the coordinates of Table 6. In some embodiments, thethree-dimensional model of the binding site on FGFR3 and/or otherstructural features are produced using the coordinates of Table 6. Usingthis model, candidate ligands that interact with the FGFR3, e.g., theFGFR3 binding site, are assessed for the desired characteristics (e.g.,interaction with FGFR3) by fitting against the model, and the result ofthe interactions is predicted. Agents predicted to be molecules capableof modulating the activity of FGFR3 can then be further screened orconfirmed against known bioassays. For example, the ability of an agentto inhibit the effects of FGFR3 can be measured using assays known inthe art. Using the modeling information and the assays described, onecan identify agents that possess FGFR3-modulating properties. Modulatorsof FGFR3 of the present disclosure can include compounds or agentshaving, for example, inhibitory activity.

Ligands which can interact with FGFR3 can also be identified usingcommercially available modeling software, such as docking programs, inwhich the solved crystal structure coordinates of Table 6 can becomputationally represented and screened against a large virtual libraryof small molecules or virtual fragment molecules for interaction with asite of interest, such as the FGFR3 binding site. Preferred smallmolecules or fragments identified in this way can be synthesized andcontacted with the FGFR3. The resulting molecular complex or associationcan be further analyzed by, for example, NMR or X-rayco-crystallography, to optimize the FGFR3 ligand interaction and/ordesired biological activity. Fragment-based drug discovery methods areknown and computational tools for their use are commercially available,for example “SAR by NMR” (Shukers, S. B., et al., Science, 1996, 274,1531-1534), “Fragments of Active Structures” (www.stromix.com; Nienaber,V. L., et al., Nat. Biotechnol., 2000, 18, 1105-1108), and “DynamicCombinatorial X-ray Crystallography” (e.g., permitting self-selection bythe protein molecule of self-assembling fragments; Congreve, M. S., etal., Angew. Chem., Int. Ed., 2003, 42, 4479-4482). Still other molecularmodeling, and like methods are discussed below and in the Examples.

In another embodiment, a candidate modulator can be identified using abiological assay such as binding to FGFR3, modulation (e.g., inhibition)of FGFR3 ligand activation of FGFR3, modulation (e.g., inhibition) ofFGFR3 biological activity. The candidate modulator can then serve as amodel to design similar agents and/or to modify the candidate modulatorfor example, to improve characteristics such as binding to FGFR3. Designor modification of candidate modulators can be accomplished using thecrystal structure coordinates and available software.

Binding Site and Other Structural Features

The present disclosure provides information inter alia about the shapeand structure of a binding site of FGFR3 in the presence of an inhibitor(anti-FGFR3 antibody). Binding sites are of significant utility infields such as drug discovery. The association of natural ligands orsubstrates with the binding sites of their corresponding receptors orenzymes is the basis of many biological mechanisms of action. Similarly,many drugs exert their biological effects through association with thebinding sites of receptors and enzymes. Such associations may occur withall or any part of the binding site. An understanding of suchassociations helps lead to the design of drugs having more favorableassociations with their target, and thus improved biological effects.Therefore, this information is valuable in designing potentialmodulators of FGFR3 binding sites, as discussed in more detail below.

The amino acid constituents of a FGFR3 binding site as defined hereinare positioned in three dimensions. The structural coordinates of FGFR3with a bound inhibitor are in Table 6. In one aspect, the structurecoordinates defining a binding site of FGFR3 include structurecoordinates of all atoms in the constituent amino acids; in anotheraspect, the structure coordinates of a binding site include structurecoordinates of just the backbone atoms of the constituent atoms. FGFR3that is bound to an inhibitor has a different conformation than wheninhibitor is not bound. In the bound state, a number of amino acidresidues form a pocket.

The FGFR3 binding site may be defined by those amino acids whosebackbone atoms are situated within about 5 Å of one or more constituentatoms of a bound ligand.

Rational Drug Design

Computational techniques can be used to screen, identify, select, designligands, and combinations thereof, capable of associating with FGFR3 orstructurally homologous molecules. Candidate modulators of FGFR3 may beidentified using functional assays, such as binding to FGFR3, and novelmodulators designed based on the structure of the candidate molecules soidentified. Knowledge of the structure coordinates for FGFR3 permits,for example, the design, the identification of synthetic compounds, andlike processes, and the design, the identification of other moleculesand like processes, that have a shape complementary to the conformationof the FGFR3 binding sites. In particular, computational techniques canbe used to identify or design ligands, such as agonists and/orantagonists, that associate with a FGFR3 binding site. Antagonists maybind to or interfere with all or a portion of an active site of FGFR3,and can be competitive, non-competitive, or uncompetitive inhibitors.Once identified and screened for biological activity, these agonists,antagonists, and combinations thereof, may be used therapeuticallyand/or prophylactically, for example, to block FGFR3 activity and thusprevent the onset and/or further progression of diseases associated withFGFR3 activity. Structure-activity data for analogues of ligands thatbind to or interfere with FGFR3 binding sites can also be obtainedcomputationally.

In some embodiments, agonists or antagonists can be designed to includecomponents that preserve and/or strengthen the interactions. Suchantagonists or agonists would include components that are able tointeract, for example, hydrogen bond with the charged amino acids foundin, e.g., either an antagonist binding site of FGFR3 (activated orunactivated, bound to substrate or unbound to substrate) or FGFR3 boundto an inhibitor or both.

In some embodiments, for FGFR3, antagonist or agonist molecules aredesigned or selected that can interact with at least one or all aminoacid residues that comprise, consist essentially of, or consist of atleast one amino acid residue corresponding to an amino acid residue inone or more of the binding site, or mixtures thereof.

Comparison of the binding site on FGFR3 to analogous sites of relatedreceptors will direct design of inhibitors that favor FGFR3 over therelated receptors. The crystal structures of other related receptors, ifthey are available can be utilized to maximize fit and/or interactionwith FGFR3 binding site and minimize the fit and/or interactions withamino acids in the corresponding positions in other receptors.

Data stored in a machine-readable storage medium that is capable ofdisplaying a graphical three-dimensional representation of the structureof FGFR3 or a structurally homologous molecule or molecular complex, asidentified herein, or portions thereof may thus be advantageously usedfor drug discovery. The structure coordinates of the ligand are used togenerate a three-dimensional image that can be computationally fit tothe three-dimensional image of FGFR3 and anti-FGFR3 antibody, or astructurally homologous molecule. The three-dimensional molecularstructure encoded by the data in the data storage medium can then becomputationally evaluated for its ability to associate with ligands.When the molecular structures encoded by the data is displayed in agraphical three-dimensional representation on a computer screen, theprotein structure can also be visually inspected for potentialassociation with ligands.

One embodiment of the method of drug design involves evaluating thepotential association of a candidate ligand with FGFR3 or a structurallyhomologous molecule or homologous complex, particularly with at leastone amino acid residue in a binding site (e.g., a binding site) of theFGFR3 or a portion of the binding site. The method of drug design thusincludes computationally evaluating the potential of a selected ligandto associate with any of the molecules or molecular complexes set forthabove. This method includes the steps of: (a) employing computationalmeans, for example, such as a programmable computer including theappropriate software known in the art or as disclosed herein, to performa fitting operation between the selected ligand and a ligand bindingsite or a subsite of the ligand binding site of the molecule ormolecular complex; and (b) analyzing the results of the fittingoperation to quantify the association between the ligand and the ligandbinding site. Optionally, the method further comprises analyzing theability of the selected ligand to interact with amino acids in the FGFR3binding site and/or subsite. The method may also further compriseoptimizing the fit of the ligand for the binding site of FGFR3 ascompared to other receptors. Optionally, the selected ligand can besynthesized, cocrystallized with FGFR3, and further modifications toselected ligand can be made to enhance inhibitory activity or fit in thebinding pocket. In addition as described previously, portions ofanti-FGFR3 antibody that bind to FGFR3 can be modified and utilized inthe method described herein. Other structural features of the FGFR3and/or FGFR3:anti-FGFR3 antibody complex can also be analyzed in thesame manner.

In another embodiment, the method of drug design involvescomputer-assisted design of ligand that associates with FGFR3, itshomologs, or portions thereof. Ligands can be designed in a step-wisefashion, one fragment at a time, or may be designed as a whole or denovo. Ligands can be designed based on the structure of molecules thatcan modulate at least one biological function of FGFR3, such asanti-FGFR3 antibody and other naturally occurring inhibitors of FGFR3.In addition, the inhibitors can be modeled on other known inhibitors ofreceptors, such as FGFRs.

In some embodiments, to be a viable drug candidate, the ligandidentified or designed according to the method must be capable ofstructurally associating with at least part of a FGFR3 binding site(e.g., a FGFR3 binding site), and must be able, sterically andenergetically, to assume a conformation that allows it to associate withthe FGFR3 binding site. Non-covalent molecular interactions important inthis association include hydrogen bonding, van der Waals interactions,hydrophobic interactions, and/or electrostatic interactions. In someembodiments, an agent may contact at least one amino acid position inthe FGFR3 binding site (e.g., a binding site) for an inhibitor, such asanti-FGFR3 antibody. Conformational considerations include the overallthree-dimensional structure and orientation of the ligand in relation tothe ligand binding site, and the spacing between various functionalgroups of a ligand that directly interact with the FGFR3 binding site orhomologs thereof.

Optionally, the potential binding of a ligand to a FGFR3 binding site isanalyzed using computer modeling techniques prior to the actualsynthesis and testing of the ligand. If these computational experimentssuggest insufficient interaction and association between it and theFGFR3 binding site, testing of the ligand is obviated. However, ifcomputer modeling indicates a strong interaction, the molecule may thenbe synthesized and tested for its ability to bind to or interfere with aFGFR3 binding site. Binding assays to determine if a compound actuallymodulates FGFR3 activity can also be performed and are well known in theart.

Several methods can be used to screen ligands or fragments for theability to associate with a FGFR3 binding site (e.g., an antagonistbinding site). This process may begin by visual inspection of, forexample, a FGFR3 binding site on the computer screen based on the FGFR3structure coordinates or other coordinates which define a similar shapegenerated from the machine-readable storage medium. Selected ligands maythen be positioned in a variety of orientations, or docked, within thebinding site. Docking may be accomplished using software such as QUANTAand SYBYL, followed by energy minimization and molecular dynamics withstandard molecular mechanics force fields, such as CHARMM and AMBER.

Specialized computer programs may also assist in the process ofselecting ligands. Examples include GRID (Hubbard, S. 1999. NatureStruct. Biol. 6:711-4); MCSS (Miranker, et al. 1991. Proteins 11:29-34)available from Molecular Simulations, San Diego, Calif.; AUTODOCK(Goodsell, et al. 1990. Proteins 8:195-202) available from ScrippsResearch Institute, La Jolla, Calif.; and DOCK (Kuntz, et al. 1982. J.Mol. Biol. 161:269-88) available from University of California, SanFrancisco, Calif.

FGFR3 binding ligands can be designed to fit a FGFR3 binding site,optionally as defined by the binding of a known modulator or oneidentified as modulating the activity of FGFR3. There are many liganddesign methods including, without limitation, LUDI (Bohm, 1992. J.Comput. Aided Molec. Design 6:61-78) available from MolecularSimulations IIIc., San Diego, Calif.; LEGEND (Nishibata, Y., and Itai,A. 1993. J. Med. Chem. 36:2921-8) available from Molecular SimulationsInc., San Diego, Calif.; LeapFrog, available from Tripos Associates, St.Louis, Mo.; and SPROUT (Gillet, et al. 1993. J. Comput. Aided Mol.Design 7:127-53) available from the University of Leeds, UK.

Once a compound has been designed or selected by the above methods, theefficiency with which that ligand may bind to or interfere with a FGFR3binding site may be tested and optimized by computational evaluation.FGFR3 binding site ligands may interact with the binding site in morethan one conformation that is similar in overall binding energy. Inthose cases, the deformation energy of binding is taken to be thedifference between the free energy of the ligand and the average energyof the conformations observed when the ligand binds to the protein.

A ligand designed or selected as binding to or interfering with a FGFR3binding site may be further computationally optimized so that in itsbound state it would preferably lack repulsive electrostatic interactionwith the target enzyme and with the surrounding water molecules. Suchnon-complementary electrostatic interactions include repulsivecharge-charge, dipole-dipole, and charge-dipole interactions.

Specific computer software is available to evaluate compound deformationenergy and electrostatic interactions. Examples of programs designed forsuch uses include: Gaussian 94, revision C (M. J. Frisch, Gaussian,Inc., Pittsburgh, Pa.); AMBER, version 4.1 (P. A. Kollman, University ofCalifornia at San Francisco,); QUANTA/CHARMM (Molecular Simulations,Inc., San Diego, Calif.); Insight II/Discover (Molecular Simulations,Inc., San Diego, Calif.); DelPhi (Molecular Simulations, Inc., SanDiego, Calif.); and AMSOL (Quantum Chemistry Program Exchange, IndianaUniversity). These programs can be implemented, for instance, using aSilicon Graphics workstation, such as an Indigo2 with IMPACT graphics.Other hardware systems and software packages will be known to thoseskilled in the art.

Another approach encompassed by this disclosure is the computationalscreening of small molecule databases for ligands or compounds that canbind in whole, or in part, to a FGFR3 binding site whether in bound orunbound conformation. In this screening, the quality of fit of suchligands to the binding site may be judged either by shapecomplementarity or by estimated interaction energy (Meng, et al., 1992.J. Comp. Chem., 13:505-24). In addition, these small molecule databasescan be screened for the ability to interact with the amino acids in theFGFR3 binding site as identified herein.

A compound that is identified or designed as a result of any of thesemethods can be obtained (or synthesized) and tested for its biologicalactivity, for example, binding and/or inhibition of FGFR3 activity.

Another method involves assessing agents that are antagonists oragonists of the FGFR3 receptor. A method comprises applying at least aportion of the crystallography coordinates of Table 6 to a computeralgorithm that generates a three-dimensional model of a FGFR3:anti-FGFR3antibody complex or the FGFR3 suitable for designing molecules that areantagonists or agonists and searching a molecular structure database toidentify potential antagonists or agonists. In some embodiments, aportion of the structural coordinates of Table 6 that define astructural feature, for example, all or a portion of a binding site(e.g., an antagonist binding site) for an inhibitor on FGFR3. The methodmay further comprise synthesizing or obtaining the agonist or antagonistand contacting the agonist or antagonist with the FGFR3 and selectingthe antagonist or agonist that modulates the FGFR3 activity compared toa control without the agonist or antagonists and/or selecting theantagonist or agonist that binds to the FGFR3.

A compound that is identified or designed as a result of any of thesemethods can be obtained (or synthesized) and tested for its biologicalactivity, for example, binding to FGFR3 and/or modulation of FGFR3activity.

7. Machine-Readable Storage Media

Transformation of the structure coordinates for all or a portion ofFGFR3, anti-FGFR3 antibody or the FGFR3:anti-FGFR3 antibody complex, orone of its ligand binding sites, or structurally homologous molecules asdefined below, or for the structural equivalents of any of thesemolecules or molecular complexes as defined above, intothree-dimensional graphical representations of the molecule or complexcan be conveniently achieved through the use of commercially-availablesoftware.

The disclosure thus further provides a machine-readable storage mediumincluding a data storage material encoded with machine-readable datawherein a machine programmed with instructions for using said datadisplays a graphical three-dimensional representation of any of themolecule or molecular complexes of this disclosure that have beendescribed above. In a preferred embodiment, the machine-readable datastorage medium includes a data storage material encoded withmachine-readable data wherein a machine programmed with instructions forusing the abovementioned data displays a graphical three-dimensionalrepresentation of a molecule or molecular complex including all or anyparts of an unbound FGFR3, a FGFR3 ligand binding site for an inhibitoror pseudo substrate, or FGFR3-like ligand binding site, anti-FGFR3antibody, FGFR3:anti-FGFR3 antibody complex as defined above. In anotherpreferred embodiment, the machine-readable data storage medium includesa data storage material encoded with machine readable data wherein amachine programmed with instructions for using the data displays agraphical three-dimensional representation of a molecule or molecularcomplex±a root mean square deviation from the atoms of the amino acidsof not more than 0.05 Å.

In an alternative embodiment, the machine-readable data storage mediumincludes a data storage material encoded with a first set of machinereadable data which includes the Fourier transform of structurecoordinates, and wherein a machine programmed with instructions forusing the data is combined with a second set of machine readable dataincluding the X-ray diffraction pattern of a molecule or molecularcomplex to determine at least a portion of the structure coordinatescorresponding to the second set of machine readable data.

For example, a system for reading a data storage medium may include acomputer including a central processing unit (“CPU”), a working memorywhich may be, for example, RAM (random access memory) or “core” memory,mass storage memory (such as one or more disk drives or CD-ROM drives),one or more display devices (e.g., cathode-ray tube (“CRT”) displays,light emitting diode (“LED”) displays, liquid crystal displays (“LCDs”),electroluminescent displays, vacuum fluorescent displays, field emissiondisplays (“FEDs”), plasma displays, projection panels, etc.), one ormore user input devices (e.g., keyboards, microphones, mice, trackballs, touch pads, etc.), one or more input lines, and one or moreoutput lines, all of which are interconnected by a conventionalbidirectional system bus. The system may be a stand-alone computer, ormay be networked (e.g., through local area networks, wide area networks,intranets, extranets, or the internet) to other systems (e.g.,computers, hosts, servers, etc.). The system may also include additionalcomputer controlled devices such as consumer electronics and appliances.

Input hardware may be coupled to the computer by input lines and may beimplemented in a variety of ways. Machine-readable data of thisdisclosure may be inputted via the use of a modem or modems connected bya telephone line or dedicated data line. Alternatively or additionally,the input hardware may include CD-ROM drives or disk drives. Inconjunction with a display terminal, a keyboard may also be used as aninput device.

Output hardware may be coupled to the computer by output lines and maysimilarly be implemented by conventional devices. By way of example, theoutput hardware may include a display device for displaying a graphicalrepresentation of a binding site of this disclosure using a program suchas QUANTA as described herein. Output hardware might also include aprinter, so that hard copy output may be produced, or a disk drive, tostore system output for later use.

In operation, a CPU coordinates the use of the various input and outputdevices, coordinates data accesses from mass storage devices, accessesto and from working memory, and determines the sequence of dataprocessing steps. A number of programs may be used to process themachine-readable data of this disclosure. Such programs are discussed inreference to the computational methods of drug discovery as describedherein. References to components of the hardware system are included asappropriate throughout the following description of the data storagemedium.

Machine-readable storage devices useful in the present disclosureinclude, but are not limited to, magnetic devices, electrical devices,optical devices, and combinations thereof. Examples of such data storagedevices include, but are not limited to, hard disk devices, CD devices,digital video disk devices, floppy disk devices, removable hard diskdevices, magneto-optic disk devices, magnetic tape devices, flash memorydevices, bubble memory devices, holographic storage devices, and anyother mass storage peripheral device. It should be understood that thesestorage devices include necessary hardware (e.g., drives, controllers,power supplies, etc.) as well as any necessary media (e.g., disks, flashcards, etc.) to enable the storage of data.

8. Therapeutic Use

FGFR3 modulator compounds obtained by methods of the invention areuseful in a variety of therapeutic settings. For example, FGFR3antagonists designed or identified using the crystal structure of FGFR3complex can be used to treat disorders or conditions where inhibition orprevention of FGFR3 binding or activity is indicated.

Likewise, FGFR3 agonists designed or identified using the binding siteand/or crystal structures provided herein can be used to treat disordersor conditions where induction or stimulation of FGFR3 is indicated.

An indication can be, for example, inhibition or stimulation of FGFR3activation and the concomitant activation of a complex set ofintracellular pathways that lead to cell growth in a variety of celltypes. Yet another indication can be, for example, in inhibition orstimulation of the FGFR3 signaling pathway. Still yet another indicationcan be, for example, in inhibition or stimulation of invasive tumorgrowth and metastasis.

The following are examples of the methods and compositions of theinvention. It is understood that various other embodiments may bepracticed, given the general description provided above.

EXAMPLES Materials and Methods Cell Lines and Cell Culture

The cell line RT4 was obtained from American Type Cell CultureCollection. Cell lines RT112, OPM2 and Ba/F3 were purchased from GermanCollection of Microorganisms and Cell Cultures (DSMZ, (Germany)).Multiple myeloma cell line KMS11 was kindly provided by Dr. TakemiOtsuki at Kawasaki Medical School (Japan). Bladder cancer cell lineTCC-97-7 was a generous gift from Dr. Margaret Knowles at St James'sUniversity Hospital (Leeds, UK). UMUC-14 cell line was obtained from Dr.H. B. Grossman (currently at University of Texas M.D. Anderson CancerCenter, TX). The cells were maintained with RPMI medium supplementedwith 10% fetal bovine serum (FBS) (Sigma), 100 U/ml penicillin, 0.1mg/ml streptomycin and L-glutamine under conditions of 5% CO₂ at 37° C.

FGFR3^(S249C) Dimerization Studies

UMUC-14 cells were grown in cysteine-free medium, treated with R3Mab orDTNB for 3 hr, and cell lysates were subject to immunoblot analysisunder reducing or non-reducing conditions. For in vitro dimerizationstudies, FGFR3-IIIb^(S249C) (residues 143-374) was cloned into pAcGP67Avector and expressed in T.ni Pro cells. The recombinant protein waspurified through Ni-NTA column followed by Superdex 5200 column. DimericFGFR3^(S249C) was eluted in 25 mM Tris (pH 7.5) and 300 mM NaCl. R3Mab(1 μM) was incubated with FGFR3^(S249C) dimer (0.1 μM) at 37° C. underthe following conditions: 100 mM KH₂PO4 (pH 7.5), 25 μM DTT, 1 mM EDTAand 0.75 mg/ml BSA. Aliquots of the reaction were taken at indicatedtime points and the reaction was stopped by adding sample buffer withoutβ-mercaptoethanol. Dimer-monomer was analyzed by immunoblot.

Xenograft Studies

All studies were approved by Genentech's Institutional Animal Care andUse Committee. Female nu/nu mice or CB17 severe combinedimmunodeficiency (SCID) mice, 6-8 weeks of age, were purchased fromCharles River Laboratory (Hollister, Calif.). Female athymic nude micewere obtained from the National Cancer Institute-Frederick CancerCenter. Mice were maintained under specific pathogen-free conditions.RT112 shRNA stable cells (7×10⁶), RT112 (7×10⁶), Ba/F3-FGFR3^(S249C)(5×10⁶), OPM2 (15×10⁶), or KMS11 cells (20×10⁶) were implantedsubcutaneously into the flank of mice in a volume of 0.2 ml inHBSS/matrigel (1:1 v/v, BD Biosciences). UMUC-14 cells (5×10⁶) wereimplanted without matrigel. Tumors were measured twice weekly using acaliper, and tumor volume was calculated using the formula: V=0.5 a×b²,where a and b are the length and width of the tumor, respectively. Whenthe mean tumor volume reached 150-200 mm³, mice were randomized intogroups of 10 and were treated twice weekly with intraperitoneal (i.p)injection of R3Mab (0.3-50 mg/kg), or a control human IgG1 diluted inHBSS. Control animals were given vehicle (HBSS) alone.

Statistics

Pooled data are expressed as mean+/−SEM. Unpaired Student's t tests(2-tailed) were used for comparison between two groups. A value ofP<0.05 was considered statistically significant in all experiments.

Generation of FGFR3 shRNA Stable Cells

Three independent FGFR3 shRNA were cloned into pHUSH vector as described(50). The sequence for FGFR3 shRNAs used in the studies is as follows:

shRNA2: (SEQ ID NO: 192)5′GATCCCCGCATCAAGCTGCGGCATCATTCAAGAGATGATGCCGCAGCT TGATGCTTTTTTGGAAA;shRNA4: (SEQ ID NO: 193)5′-GATCCCCTGCACAACCTCGACTACTATTCAAGAGATAGTAGTCGAGGTTGTGCATTTTTTGGAAA-3′; shRNA6: (SEQ ID NO: 194)5′-GATCCCCAACCTCGACTACTACAAGATTCAAGAGATCTTGTAGTAGTCGAGGTTTTTTTTGGAAA-3′.All constructs were confirmed by sequencing. EGFP control shRNA wasdescribed in our previous study (50). The shRNA containing retroviruswas produced by co-transfecting GP2-293 packaging cells (ClontechLaboratories, Mountain View, Calif.) with VSV-G (Clontech Laboratories)and pHUSH-FGFR3 shRNA constructs, and viral supernatants were harvested72 hr after transfection, and cleared of cell debris by centrifugationfor transduction experiment.

RT112 cells were maintained in RPMI 1640 medium containingtetracycline-free FBS (Clontech Laboratories), and transduced withretroviral supernatant in the presence of 4 μg/ml polybrene. 72 hoursafter infection, 2 μg/ml puromycin (Clontech Laboratories) was added tothe medium to select stable clones expressing shRNA. Stable cells wereisolated, treated with 0.1 or 1 μg/ml doxycycline (ClontechLaboratories) for 4 days, and inducible knockdown of FGFR3 proteinexpression was assessed by Western blotting analysis. Cell cycleanalyses were performed as described (51).

Selecting Phage Antibodies Specific for FGFR3

Human phage antibody libraries with synthetic diversities in theselected complementary determining regions (H1, H2, H3, L3), mimickingthe natural diversity of human IgG repertoire were used for panning. TheFab fragments were displayed bivalently on the surface of M13bacteriophage particles (52). His-tagged IgD2-D3 of human FGFR3-IIIb andMc were used as antigens. 96-well MaxiSorp immunoplates (Nunc) werecoated overnight at 4° C. with FGFR3-IIIb-His protein or FGFR3-IIIC-Hisprotein (10 μg/ml) and blocked for 1 hour with PBST buffer (PBS with0.05% Tween 20) supplemented with 1% BSA. The antibody phage librarieswere added and incubated overnight at room temperature (RT). The plateswere washed with PBST buffer and bound phage were eluted with 50 mM HCland 500 mM NaCl for 30 minutes and neutralized with equal volume of 1MTris base. Recovered phages were amplified in E. coli XL-1 blue cells.During subsequent selection rounds, the incubation time of the phageantibodies was decreased to 2 hours and the stringency of plate washingwas gradually increased (53). Unique and specific phage antibodies thatbind to both Mb and IIIc isoforms of FGFR3 were identified by phageELISA and DNA sequencing. Out of 400 clones screened, four were selectedto reformat to full length IgGs by cloning VL and VH regions ofindividual clones into LPG3 and LPG4 vectors, respectively, transientlyexpressed in mammalian cells, and purified with protein A columns (54).Clone 184.6 was selected for affinity maturation.

For affinity maturation, phagemid displaying monovalent Fab on thesurface of M13 bacteriophage (52) served as the library template forgrafting light chain (VL) and heavy chain (VH) variable domains of thephage Ab. Stop codons was incorporated in CDR-L3. A soft randomizationstrategy was adopted for affinity maturation as described (53). Twodifferent combinations of CDR loops, H1/H2/L3, H3/L3, or L1/L2/L3 wereselected for randomization. For selecting affinity-matured clones, phagelibraries were sorted against FGFR3-IIIb or IIIc-His protein, subjectedto plate sorting for the first round and followed by four rounds ofsolution phase sorting as described (52). After five rounds of panning,a high-throughput single-point competitive phage ELISA was used torapidly screen for high-affinity clones as described (55). Clones withlow ratio of the absorbance at 450 nm in the presence of 10 nM FGFR3-Histo that in the absence of FGFR3-His were chosen for furthercharacterization.

Clones 184.6.1, 184.6.21, 184.6.49, 184.6.51, 184.6.58, 184.6.62 and184.6.92 significantly reduced viability of Ba/F3-FGFR3-IIIb,Ba/F3-FGFR3-IIIc and Ba/F3-FGFR3-S249C cell lines, and clone 184.6.52significantly reduced the viability of the Ba/F3-FGFR3-S249C cell line.The increased inhibitory activity ranged from about 50-fold (clone184.6.52) to about 100-fold (clones 184.6.1, 184.6.21, 184.6.49,184.6.51, 184.6.58, 184.6.62 and 184.6.92) greater than parent clone184.6, depending on the cell line assayed. Binding kinetics of clones184.6.1, 184.6.58, and 184.6.62 to FGFR3-IIIb and FGFR3-IIIc weredetermined using BIAcore as follows:

FGFR3-IIIb KD (M) FGFR3-IIIc KD (M) 184.6 3.80E−08 1.10E−07 184.6.12.64E−10 1.44E−09 184.6.58 1.90E−10 8.80E−10 184.6.62 1.20E−10 2.24E−09Clones 184.6.1, 184.6.58, and 184.6.62 also showed improved inhibitionof FGFR3 downstream signaling in Ba/F3-FGFR3 cells, RT112 cells and OPM2cells.

Clone 184.6.1 was selected. A sequence modification, N54S, wasintroduced into HVR H2 at residue 54, to improve manufacturability,creating clone 184.6.1N54S. Clones 184.6.1 and 184.6.1N54S displayedcomparable binding kinetics (measured in Biacore assays) and comparableactivity in the Ba/F3 cell viability assay. Additional HVR H2 variantswere generated: N54S was introduced in clone 184.6.58, and N54G, N54A,or N54Q were introduced in clone 184.6.1 and 184.6.58. These clonesshowed comparable activity in the Ba/F3 cell viability assay to parentclones 184.6.1 or 184.6.58.

Another sequence modification, D30E, was introduced into HVR L1 of clone184.6.1N54S, creating clone 184.6.1NSD30E. Clone 184.6.1NSD30E and clone184.6.1N54S showed comparable binding kinetics and comparable activityin the BA/F3 cell viability assay to parent clones 184.6.1 or 184.6.58.

As used herein, “R3Mab” refers to anti-FGFR3 antibody clones184.6.1N54S, 184.6.1, or 184.6. Clone 184.6.1N54S was used in figuresand experiments referencing “R3Mab”, except in the experiments leadingto the results shown in the following figures (for which the antibodyused is shown in parentheses): FIGS. 9B (clone 184.6.1), 10 (clone184.6), 11A and B (clone 184.6), 13 (clone 184.6.1), 14A (clone184.6.1), 14B, G, and H (clone 184.6), 19 (clone 184.6.1), and 22B and C(clone 184.6.1).

BIAcore/Surface Plasmon Resonance (SRP) Analysis to Determine AntibodyBinding Affinities

Binding affinities of R3Mab to FGFR3 were measured by Biacore/SRP usinga BIAcore™-3000 instrument as described (52) with the followingmodifications. R3Mab was directly coated on CM5 biosensor chips toachieve approximately 400 response units (RU). For kinetic measurement,two-fold serial dilutions of FGFR3-IIIb or IIIc-His protein (startingfrom 67 nM) were injected in PBST buffer at 25° C. with a flow rate of30 μl/minute. Association rates (Kon, per mol/s) and dissociation rates(Koff, per s) were calculated using a simple one-one Langmuir bindingmodel (BIAcore Evaluation Software version 3.2). The equilibriumdissociation constant (Kd, per mol) was calculated as the ratio ofKoff/Kon.

Binding affinities of mouse hybridoma antibodies to FGFR3 were measuredby Biacore/SRP as follows. Human FGFR3-IIIb or Mc was coupled onto threedifferent flow cells (FC), FC2, FC3 and FC4, of a BIACORE™ CM5 sensorchip to achieve the response unit (RU) about 50 RU. Immobilization wasachieved by random coupling through amino groups using a protocolprovided by the manufacturer. Sensorgrams were recorded for binding ofhybridoma-derived anti-FGFR3 murine IgG or the Fab fragment to thesesurfaces at 25° C. by injection of a series of solutions ranging from250 nM to 0.48 nM in 2-fold increments at a flow rate of 300 min.Between each injection, 10 mM Glycine-HCl pH 1.7 was served as thebuffer to regenerate the sensor chip. The signal from the reference cell(FC1) was subtracted from the observed sensorgram at FC2, FC3 and FC4.Kinetic constants were calculated by nonlinear regression fitting of thedata according to a 1:1 Langmuir binding model using BIAcore evaluationsoftware (version 3.2) supplied by the manufacturer.

ELISA Binding Studies

cDNAs encoding the extracellular domains (ECD) of human FGFR1-IIIb, Inc,FGFR2-IIIb and Mc, FGFR3-IIIb and Mc, and FGFR4 were cloned intopRK-based vector to generate human FGFR-human Fc chimeric proteins. Therecombinant proteins were produced by transiently transfecting Chinesehamster ovary (CHO) cells and purified via protein A affinitychromatography. To test binding of antibodies to human FGFRs, Maxisorp96-well plates (Nunc) were coated overnight at 4° C. with 50 μl of 2μg/ml of FGFR ECD-human Fc chimeric proteins. After blocking withphosphate-buffered saline (PBS)/3% BSA, FGFR3 antibody was added andincubated at RT for 2 hours. Specifically bound FGFR3 antibody wasdetected using an HRP-conjugated anti-human Fab and the TMB peroxidasecolorigenic substrate (KPL, Gaithersburg, Md.).

To test the effect of antibodies to FGFR3 on FGF/FGFR3 interaction,FGFR3-Fc chimeric proteins were captured on Maxisorp plate coated withanti-human immunoglobulin Fcγ fragment-specific antibody (JacksonImmunoresearch, West Grove, Pa.). After wash, increasing amount of FGFR3antibody was added to the plate and incubated for 30 minutes. Then, FGF1or FGF9 and heparin were added for incubation at RT for 2 hours. Theplates were washed and incubated for 1 hour with biotinylatedFGF1-specific polyclonal antibody (BAF232) or biotinylated FGF9 antibody(BAF273, R&D Systems), followed by detection with streptavidin-HRP andTMB.

Generation of Ba/F3-FGFR3 Stable Cells cDNA encoding full-length humanFGFR3-IIIb or IIIc was cloned into pQCXIP vector (Clontech Laboratories,Mountain View, Calif.) to generate pQCXIP-FGFR3-IIIb or Mc. Specificmutations, i.e., R248C, S249C, G372C, Y375C and K652E, were introducedinto the cDNA via QuickChange (Stratagene, La Jolla, Calif.). Togenerate Ba/F3 stable cells expressing wild type or mutant FGFR3,various pQCXIP-FGFR3 constructs were co-transfected into packaging cellsGP2-293 with VSV-G plasmid (Clontech Laboratories). After selection with2 μg/ml puromycin for two weeks, cells expressing wild type or mutantFGFR3 were stained with Phycoerythrin-conjugated anti-human FGFR3 mAb(FAB766P, R&D Systems), and selected through fluorescence-activated cellsorting (FACS) for functional assays. For cell proliferation assay in96-well micro-titer plate, the following cell density was used: Forcells expressing wild type FGFR3-IIIb and FGFR3-K652E: 5,000 cells/well;for the rest: 10,000 cells/well. Cells were seeded in RPMI 1640 mediumsupplemented with 10% fetal bovine serum, 10 ng/ml FGF1 plus 10 μg/mlheparin (Sigma-Aldrich, St. Louis, Mo.). R3Mab was added at indicatedconcentration and mouse hybridoma FGFR3 antibodies were added at 2000 to0.49 ng/ml (in four-fold serial dilutions) in the FGFR3-IIIb experimentand 5000 to 1.2 ng/ml (in four-fold serial dilutions) in the FGFR3-IIIcexperiment. After incubation for 72 hours, cell viability was assessedwith CellTiter-Glo (Promega, Madison, Wis.).

Cell Proliferation Assay

For proliferation assays for RT112, RT4 and TCC-97-7 cells, 3000cells/well were seeded into 96-well micro-titer plate and were allowedto adhere overnight. The medium was then replaced with low serum medium(0.5% FBS) with control or R3Mab at concentrations indicated. Following4 days incubation, 1 μCi of [Methyl-³H] thymidine (PerkinElmer, Waltham,Mass.) was added to each well, and incubated for additional 16 hours.Cells were transferred to UniFilters using Packard Filtermate Harvester,and [³H]-thymidine incorporated into the genomic DNA of growing cellswas measured using TopCount (PerkinElmer). In some cases, cell viabilitywas assessed with CellTiter-Glo (Promega) following incubation withantibodies for 4 days. Values are presented as means+/−SE ofquadruplets.

Clonal Growth Assay

The effect of R3Mab on cell clonogenicity was assessed following apreviously described protocol (50). In brief, 400 UMUC-14 cells wereseeded into 6-well plate in DMEM medium supplemented with 10% fetalbovine serum to allow adhesion overnight. Then R3Mab or control antibodydiluted in 0.1% BSA medium was added to a final concentration of 10μg/ml. Equal volume of 0.1% BSA medium alone (Mock) was used as anothercontrol. The cells were incubated for about 12 days until cells incontrol groups formed sufficiently large colonies. Colonies were stainedwith 0.5% crystal violet, and the number and size of colonies werequantitated using GelCount (Oxford, UK). The number of colonies largerthan 120 μm in diameter was presented as mean+/−SEM (n=12).

Immunoprecipitation and Immunoblotting Analyses

To study the effect of antibodies on FGFR3 signaling, cells were starvedin serum-free medium overnight prior to the beginning of treatment.Cells were incubated with either antibodies diluted in 0.1% BSA (w/v),RPMI 1640 medium, or with 0.1% BSA medium alone (Mock). After 3 hours at37° C., FGF1 (final concentration of 15 ng/ml) and heparin (finalconcentration of 5-10 μg/ml) were added to half of the samples. Ascontrols, a similar volume of heparin alone was added to the other halfof samples. The incubation was continued for 10 min. Supernatants wereremoved by aspiration, and cells were washed with ice-cold PBS, thenlysed in RIPA buffer (Upstate, Charlottesville, Va.) supplemented with 1mM sodium orthovanadate and Complete protease inhibitor cocktail (RocheApplied Science, Indianapolis, Ind.). The lysates were cleared ofinsoluble materials by centrifugation.

FGFR3 was immunoprecipitated using a rabbit polyclonal antibody (sc-123,Santa Cruz Biotechnology, Santa Cruz, Calif.) and analyzed by sodiumdodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.Phosphorylated FGFR3 was assessed with a monoclonal antibody againstphospho-tyrosine (4G10, Upstate). Total FGFR3 was probed with amonoclonal antibody against FGFR3 (sc-13121, Santa Cruz Biotechnology).Phosphorylation and activation of FGFR3 signaling pathway were probedusing the following antibodies: anti-FGFR^(Y653/654), anti-FRS2α^(Y196),anti-phospho-p44/42 MAPK^(T202/Y204), anti-total p44/42 MAPK andanti-AKT^(S473) were obtained from Cell Signaling Technology (Danvers,Mass.); and anti-total FRS2α (sc-8318) was purchased from Santa CruzBiotechnology (Santa Cruz, Calif.). The blots were visualized using achemiluminescent substrate (ECL Plus, Amersham Pharmacia Biotech,Piscataway, N.J.).

Antibody Epitope Mapping

To determine the epitope of R3Mab, 13 overlapping peptides, each of 15amino acids in length, were synthesized to cover the extracellulardomain of human FGFR3 from residues 138 to 310. The peptides werebiotinylated at the C-terminus, and captured on streptavidin plates(Pierce, Rockford, Ill.) overnight. After blocking with PBS/3% BSA, theplates were incubated with R3Mab and detected using an HRP-conjugatedanti-human IgG (Jackson Immunoresearch) and the TMB peroxidasecolorigenic substrate (KPL, Gaithersburg, Md.).

Mouse anti-human FGFR3 hybridoma antibodies 1G6, 6G1, and 15B2 weretested in ELISA assay to identify their binding epitopes. 1G6, 6G1 and15B2 bind to human FGFR IgD2-IgD3 (both IIIb and IIIc isoforms), whereas5B8 only binds IgD2-IgD3 of human FGFR3-IIIb. In a competition assay,1G6, 6G1 and 15B2 competed with each other to bind human FGFR3,suggesting that 1G6, 6G1 and 15B2 have overlapping epitopes. None of thehybridoma antibodies competed with phage antibody 184.6, suggesting thatthe hybridoma antibodies have distinct epitope(s) from 184.6.

Preparation and Molecular Cloning of Mouse Anti-FGFR3 Antibodies 1G6,6G1, and 15B2

BALB/c mice were immunized 12 times with 2.0 μg of FGFR3-IIIb (rhFGFR3(IIIB)/Fc Chimera, from R&D Systems, catalog #1264-FR, lot # CYHO25011,or with 2.0 μg of FGFR3-IIIc (rhFGFR3 (IIIc)/Fc Chimera, from R&DSystems, catalog #766-FR, lot # CWZ055041, resuspended in monophosphoryllipid A/trehalose dicorynomycolate adjuvant (Corixa, Hamilton, Mont.)into each hind footpad twice a week. Three days after final boost,popliteal lymph nodes were fused with mouse myeloma cell lineP3X63Ag.U.1, via electrofusion (Hybrimune, Cyto Pulse Sciences, GlenBurnie, Md.). Fused hybridoma cells were selected from unfused poplitealnode or myeloma cells using hypoxanthin-aminopterin-thymidine (HAT)selection in Medium D from the ClonaCell® hybridoma selection kit(StemCell Technologies, Inc., Vancouver, BC, Canada). Culturesupernatants were initially screened for its ability to bind toFGFR3-IIIb and FGFR3-IIIc by ELISA, and hybridomas of interest weresubsequently screened for its ability to stain by FACS on transfectedFGFR3-IIIb Ba/F cells and control Ba/F, as well as antibody blockingactivity. Selected hybridomas were then cloned by limiting dilution.

Total RNA was extracted from hybridoma cells producing the mouse antihuman FGFRIII monoclonal antibody 1G6 and 15B2, using RNeasy Mini Kit(Qiagen, Germany). The variable light (VL) and variable heavy (VH)domains were amplified using RT-PCR with the following degenerateprimers:

1G6: Light chain (LC) forward: (SEQ ID NO: 195)5′-GTCAGATATCGTKCTSACMCARTCTCCWGC-3′ Heavy chain (HC) forward:(SEQ ID NO: 196) 5′-GATCGACGTACGCTGAGATCCARYTGCARCARTCTGG-3′ 6G1:Light chain (LC) forward: (SEQ ID NO: 197)5′-GTCAGATATCGTGCTGACMCARTCTCC-3′ Heavy chain (HC) forward:(SEQ ID NO: 198) 5′-GATCGACGTACGCTGAGATCCARYTGCARCARTCTGG-3′ 15B2:Light chain (LC) forward: (SEQ ID NO: 199)5′-GTACGATATCCAGATGACMCARTCTCC-3′ Heavy chain (HC) forward:(SEQ ID NO: 200) 5′-GATCGACGTACGCTGAGATCCARYTGCARCARTCTGG-3′

Light chain and Heavy chain reverse primer for all three clones are asfollowed:

Light chain reverse: (SEQ ID NO: 201) 5′-TTTDAKYTCCAGCTTGGTACC-3′Heavy chain reverse: (SEQ ID NO: 202)5′-ACAGTGGGCCCTTGGTGGAGGCTGMRGAGACDGTGASHRDRGT-3′.

The forward primers were specific for the N-terminal amino acid sequenceof the VL and VH region. The LC and HC reverse primers were designed toanneal to a region in the constant light (CL) and constant heavy domain1 (CH1), respectively, which are highly conserved across species.

Amplified VL was cloned into a pRK mammalian cell expression vector(Shields et al, (2000) J. Biol. Chem. 276:659) containing the humankappa constant domain. Amplified VH was inserted to a pRK mammalian cellexpression vector encoding the full-length human IgG1 constant domain.The sequence of the heavy and light chains was determined usingconventional methods.

Crystallization, Structure Determination and Refinement

The human FGFR3-IIIb ECD (residues 143-374) was cloned into pAcGP67Avector (BD Bioscience, San Jose, Calif.), produced in T.ni Pro cells andpurified using Ni-NTA column followed by size exclusion chromatography.The R3Mab Fab was expressed in E. coli and purified sequentially over aprotein G affinity column, an SP sepharose column and a Superdex 75column. The R3Mab Fab had the following sequence:

Light chain: (SEQ ID NO: 276)DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC, andHeavy chain: (SEQ ID NO: 277)EVQLVESGGGLVQPGGSLRLSCAASGFTFTSTGISWVRQAPGKGLEWVGRIY PTNGSTNYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARARTYGIYDLYVDYTEYVMDYWGQGTLVASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH

Fab-FGFR3 complex was generated by incubating the Fab with an excess ofFGFR3 ECD, and the complex was then deglycosylated and purified over aSuperdex-200 sizing column in 20 mM TrisCl pH 7.5 and 200 mM NaClbuffer. The complex-containing fractions were pooled and concentrated to20 mg/ml and used in crystallization trials. Crystals used in thestructure determination were grown at 4° C. from the followingcondition: 0.1 M sodium cacodylate pH 6.5, 40% MPD and 5% PEG8000 usingvapor diffusion method. Data was processed using HKL2000 and Scalepack(56). The structure was solved with molecular replacement using programPhaser (57) and the coordinates of 1RY3 (FGFR3) and 1N8Z (Fab-fragment).The model was completed using program Coot (58) and the structurerefined to R/R_(free) of 20.4%/24.3% with program Refmac (59).Coordinates and structure factors were deposited in the Protein DataBank with accession code 3GRW and are also disclosed in U.S. Ser. No.61/163,222, filed on Mar. 25, 2009, the contents of which is herebyincorporated by reference.

ADCC Assay

Human PBMCs were isolated by Ficoll gradient centrifugation ofheparinized blood, and ADCC was measured using the multiple myeloma celllines OPM2 or KMS11 or bladder cancer cell lines RT112 or UMUC-14 astarget and PBMCs as effector cells at a 1:100 target:effector ratio. Thetarget cells (10,000 cells/well) were treated with R3Mab or with controlhuman IgG1 for 4 hours at 37° C. Cytotoxicity was determined bymeasuring LDH release using the CytoTox-ONE Homogeneous MembraneIntegrity Assay following manufacturer's instructions (Promega, Madison,Wis.). The results are expressed as percentage of specific cytolysisusing the formula: Cytotoxicity (%)=[(Experimental lysis−Experimentalspontaneous lysis)/(Target maximum lysis−target spontaneous lysis)]×100,where spontaneous lysis is the nonspecific cytolysis in the absence ofantibody, and target maximum lysis is induced by 1% Triton X-100.

Results

Inducible shRNA Knockdown of FGFR3 Attenuates Bladder Cancer Growth InVivo

As a prelude to assessing the importance of FGFR3 for bladder tumorgrowth in vivo, we examined the effect of FGFR3 knockdown in vitro.Several FGFR3 small interfering (si) RNAs effectively downregulatedFGFR3 in bladder cancer cell lines expressing either WT (RT112, RT4,SW780) or mutant (UMUC-14, S249C mutation) FGFR3. FGFR3 knockdown in allfour cell lines markedly suppressed proliferation in culture (FIG. 15).Next, we generated stable RT112 cell lines expressingdoxycycline-inducible FGFR3 shRNA. Induction of three independent FGFR3shRNAs by doxycycline diminished FGFR3 expression, whereas induction ofa control shRNA targeting EGFP had no effect (FIG. 7A). In the absenceof exogenous FGF, doxycycline treatment reduced [³H]-thymidineincorporation by cells expressing different FGFR3 shRNAs, but notcontrol shRNA (FIG. 7B), confirming that FGFR3 knockdown inhibitsproliferation. Further analysis of exponentially growing RT112 cellsrevealed that FGFR3 knockdown over a 72 hr treatment with doxycyclinemarkedly and specifically reduced the percentage of cells in the S andG2 phases of the cell cycle, with a concomitant increase of cells in G1phase (FIG. 7C). Similar effect was observed with two other FGFR3 shRNAs(FIG. 16A). No significant numbers of cells with a sub-diploid DNAcontent were detected, suggesting no change in apoptosis levels. Hence,the inhibitory effect of FGFR3 knockdown on the proliferation of RT112cells is mainly due to attenuation of cell cycle progression.

We next evaluated the effect of FGFR3 knockdown on the growth ofpre-established RT112 tumor xenografts in mice. FGFR3 knockdownsubstantially and specifically suppressed tumor growth (FIG. 7D, toppanels and FIG. 16B). Analysis of day 45 tumor samples confirmedeffective FGFR3 knockdown upon doxycycline induction of FGFR3 shRNA ascompared to control shRNA (FIG. 7D, bottom panels). These resultsdemonstrate that FGFR3 is critically important both in vitro and in vivofor the growth of RT bladder cancer cells.

Generation of a Blocking Anti-FGFR3 Monoclonal Antibody

To examine further the importance of FGFR3 in tumor growth and toexplore the potential of this receptor as a therapeutic target, wedeveloped an antagonistic anti-FGFR3 monoclonal antibody (dubbed R3Mab)using a phage display approach. We selected this particular antibodybased on its ability to block both ligand binding and dimerization byFGFR3, and its unique capacity to inhibit not only WT FGFR3 but also themost prevalent cancer-associated mutants of this receptor (see below).R3Mab targets the extracellular IgD2 and IgD3 domains of FGFR3, whichare necessary and sufficient for FGF binding (4). R3Mab bound both theIIIb and Mc isoforms of human FGFR3, but showed no detectable binding toFGFR1, FGFR2 or FGFR4 (FIG. 8A). Biacore analysis indicated that R3Mabhad similar apparent affinity to murine, cynomolgus monkey and humanFGFR3-IIIc (data not shown). The affinity of R3Mab to human FGFR3 isshown in Table 2.

TABLE 2 Affinity of R3Mab to human FGFR3 determined by BIAcore analysis.R3 Ab captured on chips Human FGFR3 ECD kon/(1/Ms) koff(1/s) Kd(M) IIIb1.80E+06 2.00E−04 1.11E−10 IIIc 9.10E+04 3.20E−04 3.52E−09

We next tested the ability of R3Mab to block FGFR3 binding to FGF1 andFGF9. R3Mab strongly inhibited binding of FGF1 to FGFR3-IIIb and -IIIc,with half-maximal inhibitory concentrations (IC₅₀) of 0.3 nM and 1.7 nM,respectively (FIG. 8B,C). Similarly, R3Mab efficiently blocked FGF9binding to FGFR3-IIIb and -IIIc, with an IC₅₀ of 1.1 nM and 1.3 nM,respectively (FIG. 8D,E).

R3Mab Inhibits WT FGFR3 and its Most Prevalent Cancer-Associated MutantVariants

To examine whether R3Mab inhibits cell proliferation driven by WT ormutant FGFR3, we took advantage of the observation that ectopic FGFR3expression in murine pro-B cell Ba/F3 confers interleukin(IL)-3-independent, FGF1-dependent proliferation and survival (29). Inthe absence of FGF1 and IL-3, Ba/F3 cells stably expressing WT FGFR3were not viable, while FGF1 greatly enhanced their proliferation (FIG.9A). R3Mab specifically blocked FGF1-stimulated Ba/F3-FGFR3 cellproliferation in a dose-dependent manner (FIG. 9A). We next evaluatedthe impact of R3Mab on FGFR3 signaling in these cells. FGF1 inducedphosphorylation and activation of FGFR3 and concomitant activation ofp44/42 MAPK, while R3Mab effectively suppressed the activation of bothmolecules (FIG. 9B).

In bladder cancer, somatic activating mutations in FGFR3 cluster withinthe linker region between IgD2 and IgD3, the extracellular juxtamembranedomain, or the kinase domain (FIG. 9C). The extracellular missensesubstitutions most often give rise to an unpaired cysteine, leading toligand-independent dimerization of FGFR3. These mutations cause markedlydifferent levels of constitutive FGFR3 activation, possibly owing to adifferential impact on the orientation of the cytoplasmic kinase domain(30, 31). The most frequent mutations are S249C, Y375C, R248C, G372C,and K652E, which together account for 98% of all FGFR3 mutations inbladder cancer (32). We reasoned that an optimal therapeutic agentshould block not only the WT FGFR3 protein, which is overexpressed incertain cancers, but also the most prevalent tumor-associated FGFR3mutants. To assess R3Mab further, we generated Ba/F3 cell lines stablyexpressing each of the five most common FGFR3 mutant variants. Allmutants were expressed at similar levels at the cell surface, and thecysteine mutants dimerized spontaneously without ligand (data notshown). Cell lines expressing different cysteine mutants exhibited avariable growth response to FGF1, consistent with earlier findings (30,31). As previously reported (33), cells expressing FGFR3^(R248C)displayed constitutive, ligand-independent proliferation, and were notresponsive to FGF1 (FIG. 9D). Similarly, the most frequent mutation,FGFR3^(S249C), conferred ligand-independent proliferation (FIG. 9E).Remarkably, R3Mab suppressed constitutive proliferation driven by eithermutant (FIG. 9 D,E). Cells expressing the juxtamembrane domain mutationsFGFR3^(G372C) (FIG. 9F) or FGFR3^(Y375C) (FIG. 9G) required FGF1 forproliferation, and their growth was completely blocked by R3Mab. Cellsexpressing FGFR3^(K652E) showed weak ligand-independent proliferationand significant growth in response to FGF1 (33). R3Mab did not affectthe weak basal activity of FGFR3^(K652E) (data not shown), but nearlyabolished ligand-induced proliferation mediated by this mutant (FIG.9H). Hence, R3Mab has a unique capacity to inhibit both WT and prevalentcancer-associated mutants of FGFR3. Moreover, R3Mab did not displaydetectable agonist activity.

As a separate effort, we generated and characterized multiplemouse-anti-human FGFR3 hybridoma antibodies. None of the hybridomaantibodies could inhibit all the cancer-linked FGFR3 mutants we tested(FIG. 17), nor did they share overlapping epitopes with R3Mab.

Moreover, all of the hybridoma antibodies showed agonist activity,strongly stimulating proliferation of cancer-linked FGFR3 mutants R248Cand S249C, and showing some stimulation of proliferation of mutantsY375C and G370C. The hybridoma antibodies showed differential levels ofantagonist and agonism, depending on the FGFR3 mutant tested, asfollows:

1G6 6G1 15B2 FGFR3-IIIb wildtype inhibition inhibition inhibitionFGFR3-IIIb R248C 2X stimulation 4-5X stimulation 3-4X stimulationFGFR3-IIIbS249C 2X stimulation 4-5X stimulation 4-5X stimulationFGFR3-IIIb Y375C 1.2-1.5X 1.2-1.5X 1.2-1.5X stimulation stimulationstimulation FGFR3-IIIb K652E 50% inhibition 60-70% inhibition inhibitionFGFR3-IIIc inhibition inhibition inhibition FGFR3-IIIc G370C No effect20-30% inhibition 10-2-% inhibitionThus, the hybridoma antibodies showed unpredictable differential effecton Ba/F3 cells cell proliferation driven by various FGFR3 mutants.

Characterization of Mouse-Anti-Human FGFR3 Hybridoma Antibodies

Mouse anti-human FGFR3 hybridoma antibodies were further characterizedas follows:

(1) In an assay to test ability of anti-FGFR3 murine hybridomaantibodies to inhibit FGF1 binding to human FGFR3-IIIb and IIIcisoforms, antibodies 1G6, 6G1 and 15B2 were able to block binding ofFGF1 to human FGFR3-IIIb and IIIc isoforms in a dose-dependent manner.When tested across an antibody concentration range of about 2000 to 0.49ng/ml, antibodies 1G6, 6G1 and 15B2 blocked FGF1 binding to FGFR3-IIIbwith IC50 values of 0.69, 0.87 and 0.72 nM. When tested across anantibody concentration range of about 5000 to 1.2 ng/ml, antibodies 1G6,6G1 and 15B2 blocked FGF1 binding to FGFR3-IIIc with IC50 values of0.57, 3.4 and 0.7 nM, respectively.

(2) In an assay to test ability of anti-FGFR3 murine hybridomaantibodies to inhibit FGF9 binding to human FGFR3-IIIb and IIIcisoforms, antibodies 1G6, 6G1 and 15B2 efficiently blocked binding ofFGF1 to human FGFR3-IIIb and IIIc isoforms in a dose-dependent manner.When tested across an antibody concentration range of about 2000 to 0.49ng/m, antibodies 1G6, 6G1 and 15B2 blocked FGF9 binding to FGFR3-IIIbwith IC50 values of 0.13, 0.16, and 0.07 nM, respectively. When testedacross an antibody concentration range of about 5000 to 1.2 ng/ml,antibodies 1G6, 6G1 and 15B2 blocked FGF9 binding to FGFR3-IIIc withIC50 values of 0.13, 0.11, and 0.07 nM, respectively.

(3) The binding affinity of full-length anti-FGFR3 murine hybridomaantibodies 1G6, 6G1 and 15B2 was determined using Biacore analysis. Theresults of this analysis are shown in Table 3.

TABLE 3 FGFR3-IIIB FGFR3-IIIC Anti- kon koff Kd kon koff Kd body(10⁵M⁻¹s⁻¹) (10⁻⁴s⁻¹) (nM) (10⁵M⁻¹s⁻¹) (10⁻⁴s⁻¹) (nM) 1G6 2.2 3.1 1.42.2 2.8 1.3 mIgG 6G1 2.7 3.8 1.4 2.6 3.2 1.2 mIgG 15B2 4.1 29 7.1 3.5 3911.1 mIgG

(4) In an assay to test ability of anti-FGFR3 murine hybridomaantibodies to inhibit Ba/F3 cell proliferation driven by humanFGFR3-IIIb or Mc, antibodies 1G6, 6G1 and 15B2 were able to block Ba/F3cell proliferation driven by human FGFR3-IIIb or IIIc in adose-dependent manner. When tested across an antibody concentrationrange of about 0.01 to 100 ug/ml, antibodies 1G6, 6G1 and 15B2 blockedBa/F3 cell proliferation driven by FGFR3-IIIb with IC50 values of 3-5nM, 3 nM, and 6-8 nM, respectively, and blocked Ba/F3 cell proliferationdriven by FGFR3-IIIc with IC50 values of 10-35 nM, 24 nM, and 60 nM,respectively.

(5) In an assay to test ability of anti-FGFR3 murine hybridomaantibodies to inhibit FGF1-induced signaling in Ba/F3 cells expressinghuman FGFR3-IIIb, antibodies 1G6, 6G1 and 15B2 were able to blockFGF1-induced signaling in Ba/F3 cells expressing human FGFR3-IIIb in adose-dependent manner when tested across an antibody concentration rangeof about 0.25 to 6.75 ug/ml. 25 ng/ml of FGF1 was used in thisexperiment. In the absence of FGF1, antibody treatment had no effect onFGFR3 activation.

(6) In an assay to test ability of anti-FGFR3 murine hybridomaantibodies to inhibit FGF1-induced signaling in Ba/F3 cells expressinghuman FGFR3-IIIc, antibodies 1G6, 6G1 and 15B2 were able to blockFGF1-induced signaling in Ba/F3 cells expressing human FGFR3-IIIc in adose-dependent manner when tested across an antibody concentration rangeof about 0.25 to 6.75 ug/ml. 25 ng/ml of FGF1 was used in thisexperiment. In the absence of FGF1, antibody treatment had no effect onFGFR3 activation.

Structural Basis for the Interaction of R3Mab with FGFR3

To gain insight into R3Mab's mode of interaction with FGFR3, wesynthesized a panel of 13 overlapping peptides spanning the FGFR3-IIIbIgD2 and D3 regions and tested their binding to R3Mab. Peptides 3(residues 164-178) and 11 (residues 269-283) showed specific binding toR3Mab, with peptide 3 having a stronger interaction (FIG. 10A),indicating that the corresponding regions on FGFR3 are critical forrecognition by R3Mab. Previous crystallographic studies of FGFR1 incomplex with FGF2 identified critical receptor residues engaged indirect binding to FGF and heparin as well as in receptor dimerization(34). Alignment of FGFR3 peptides 3 and 11 with the functionallyimportant sites in FGFR1 revealed that these peptides encompasscorresponding FGFR1 residues essential for direct FGF2 binding, receptordimerization, as well as interaction with heparin (FIG. 10B). These dataindicate that the epitope of R3Mab on FGFR3 overlaps with receptorresidues engaged in ligand association and receptor-receptorinteraction.

We next crystallized the complex between the Fab fragment of R3Mab andthe extracellular IgD2-D3 region of human FGFR3-IIIb, and determined theX-ray structure at 2.1 Å resolution (FIG. 10 C, D; Table 4). In thiscomplex, approximately 1400 Å2 and 1500 Å2 of solvent-accessible surfaceareas are buried on FGFR3 and the Fab, respectively. About 80% of theburied interface involves IgD2, while the remainder entails the linkerand IgD3 regions. On the Fab side of the complex, about 40% of theburied interface involve complementarity-determining region (CDR)-H3,20% CDR-H2, 20% CDR-L2, and minor contributions are from other CDRs andframework residues. Notably, amino acids (AAs) from CDR-H3 form twoβ-strands, which extend the β-sheet of IgD2 (FIG. 10D). The Fabinteracts with AAs that constitute the FGF binding site of FGFR3 as wellas residues that form the receptor dimerization interface, as previouslyidentified in various dimeric FGF:FGFR complexes (e.g., PDB code 1CVS,(34); and FIG. 10C, areas in grey/crosshatched and dark grey). Theinteraction interfaces identified by crystallography were fullyconsistent with the peptide-based data (FIG. 18 A, B). Together, theseresults reveal how R3Mab inhibits ligand binding, and further suggestthat binding of R3Mab to FGFR3 may prevent receptor dimerization. FGFR3amino acids that contact R3Mab are shown in Table 5. Crystallographiccoordinates for this structure are deposited in the Protein Data Bankwith accession code 3GRW and shown in Table 6.

TABLE 5 Residues in FGFR3 that are in contact with R3Mab Residue Buriedsurface of residue in the interface THR 154 0.10 ARG 155 16.50 ARG 158105.40 MET 159 6.00 LYS 161 52.50 LYS 162 1.70 LEU 163 12.30 LEU 16455.10 ALA 165 10.10 VAL 166 10.60 PRO 167 45.50 ALA 168 22.60 ALA 16963.60 ASN 170 75.40 THR 171 83.00 VAL 172 1.70 ARG 173 91.70 PHE 1741.50 ARG 175 95.60 PRO 177 15.90 GLY 202 2.10 LYS 205 63.40 ARG 20767.60 GLN 210 31.60 SER 212 0.40 VAL 214 26.40 GLU 216 48.90 SER 2171.80 TYR 241 15.90 LEU 246 3.10 GLU 247 1.80 ARG 248 46.90 TYR 278 32.20SER 279 1.80 ASP 280 19.80 ALA 281 3.00 GLN 282 24.80 PRO 283 0.50 SER314 1.20 GLU 315 82.60 SER 316 33.20 VAL 317 56.60 GLU 318 51.50

TABLE 4 Summary of crystallographic analysis Data collection FGFR3-IIIb:R3MAb Fab Space group P2₁2₁2₁ Cell parameters a = 58.5, b = 99.3, c =143.7 Resolution (Å) 25-2.1 R_(sym) ^(a) 0.098 (0.663)^(b) Number ofobservations 288498 Unique reflections 49851 Completeness (%) 99.99(100.0)^(b) Refinement Resolution (Å) 20-2.1 Number of reflections 46714Final R^(c), R_(free) (F > 0) 0.187, 0.224 Number of non-H atoms 5270Number of Amino Acids 650 Sulfate 1 Sugar 1 Solvent atoms 274 Rmsd bonds(Å) 0.011 Rmsd angles (°) 1.4 ^(a)R_(sym) = Σ|I−<I>|/Σ I. <I> is theaverage intensity of symmetry related observations of a uniquereflection. ^(b)Numbers in parentheses refer to the highest resolutionshell. ^(c)R = Σ|F_(o)−F_(c)|/ΣF_(o). R_(free) is calculated as R, butfor 5% of the reflections excluded from all refinement.

We compared the R3Mab-FGFR3 structure with a previously publishedstructure of FGFR3-IIIc in complex with FGF1 (4, 35) (FIG. 10E, 10F).Superposition of the antibody-receptor and ligand-receptor complexesrevealed that there are no major conformational differences within theindividual receptor domains, except in the region that distinguishesFGFR3-IIIc from FGFR3-IIIb; however, the orientation of IgD3 relative toIgD2 was drastically different (FIG. 10E, white and grey; FIG. 10F,white and grey-mesh). Since the relative positions of IgD2 and IgD3 arecritical for ligand binding, the alternate conformation adopted by IgD3upon R3Mab binding may provide an additional mechanism to prevent ligandinteraction with FGFR3.

R3Mab Inhibits Endogenous WT and Mutant FGFR3 in Bladder Cancer Cells

To assess whether R3Mab could suppress FGFR3 function in bladder cancercells, we first examined RT112 and RT4 cell lines, which express WTFGFR3. R3Mab strongly inhibited [³H]-thymidine incorporation by RT112cells (FIG. 11A) and exerted a significant, though more moderatesuppression of RT4 cell proliferation (FIG. 19A). To investigate R3Mab'seffect on FGFR3 activation, we examined the phosphorylation of FGFR3 inRT112 cells. Consistent with the results in Ba/F3-FGFR3 cells (FIG. 9B),R3Mab markedly attenuated FGF1-induced FGFR3 phosphorylation (FIG. 11B).We next examined phosphorylation of FRS2α, AKT, and p44/42 MAPK, threedownstream mediators of FGFR3 signaling. FGF1 strongly activated thesemolecules in RT112 cells, while R3Mab significantly diminished thisactivation (FIG. 11B). Similarly, R3Mab suppressed FGF1-inducedphosphorylation of FGFR3 and MAPK in RT4 cells (FIG. 19B).

We next investigated whether R3Mab could inhibit activation ofendogenous mutant FGFR3 in human bladder cancer cells. S249C is the mostfrequent FGFR3 mutation in bladder cancer (FIG. 9C). Two available celllines, UMUC-14 and TCC-97-7, carry a mutated FGFR3^(S249C) allele (Ref.36 and data not shown). Although R3Mab did not affect the exponentialgrowth of UMUC-14 cells in culture (data not shown), it significantlyreduced the clonal growth of these cells (FIG. 11C). Specifically, R3Mabdecreased the number of colonies larger than 120 μm in diameterapproximately by 77% as compared with control antibody (FIG. 11D).Furthermore, R3Mab inhibited [³H]-thymidine incorporation by TCC-97-7cells in culture (FIG. 19C).

The S249C mutation is reported to result in ligand-independentactivation of FGFR3 (26, 30). Indeed, FGFR3^(S249C) was constitutivelyphosphorylated irrespective of FGF1 treatment in UMUC-14 cells andTCC-97-7 cells, while R3Mab reduced constitutive phosphorylation ofFGFR3^(S249C) as compared with control antibody in both cell lines(FIGS. 11E, 19D).

R3Mab Inhibits Dimer Formation by FGFR3^(S249C)

The ability of R3Mab to inhibit constitutive FGFR3^(S249C) signaling andproliferation in bladder cancer cells was surprising, considering thatthis mutant can undergo disulfide-linked, ligand-independentdimerization (26, 30). To explore how R3Mab inhibits FGFR3^(S249C), weexamined the effect of R3Mab on the oligomeric state of this mutant inUMUC-14 cells. Under reducing conditions, FGFR3^(S249C) migrated as asingle band of ˜97 kDa, consistent with monomeric size (FIG. 12A). Undernon-reducing conditions, in cells treated with control antibody a largefraction of FGFR3^(S249C) appeared as a band of ˜200 kDa, regardless ofFGF1 addition, indicating a constitutive dimeric state (FIG. 12A). R3Mabtreatment substantially decreased the amount of dimers, with aconcomitant increase in monomers (FIG. 12A). Consistently, R3Mabdecreased the level of FGFR3^(S249C) dimers in TCC-97-7 cellsirrespective of FGF1 treatment (FIG. 19E).

How does R3Mab decrease the FGFR3^(S249C) dimer levels in bladder cancercells? One potential explanation is that it may disrupt theFGFR3^(S249C) dimer through antibody-induced FGFR3 internalization andtrafficking through endosomes or lysosomes. We tested this possibilityby pharmacologically intervening with endocytosis. R3Mab nonethelessdecreased the amount of dimer in UMUC-14 cells pre-treated with variousendocytosis inhibitors, despite substantial blockade of FGFR3^(S249C)internalization (FIG. 20 A, B). Thus, dimer disruption by R3Mab isindependent of endocytosis. Another possible explanation is thatcellular FGFR3^(S249C) may exist in a dynamic monomer-dimer equilibrium;accordingly, binding of R3Mab to monomeric FGFR3^(S249C) could preventdimer formation and thereby shift the equilibrium toward the monomericstate. To examine this possibility, we used the non-cell-permeatingagent 5,5′Dithiobis 2-nitrobenzoic acid (DTNB), which selectively reactswith and blocks free sulfhydryl groups of unpaired cysteines (37).Treatment of UMUC-14 cells with DTNB led to the accumulation ofFGFR3^(S249C) monomers at the expense of dimers (FIG. 12B), indicatingthat FGFR3^(S249C) exists in a dynamic equilibrium between monomers anddimers.

To test whether R3Mab affects this equilibrium, we generated a solublerecombinant protein comprising the IgD2-D3 domains of FGFR3^(S249C) andisolated the dimers by size exclusion chromatography. We incubated thedimers with buffer or antibodies in the presence of a very lowconcentration of reducing agent (25 μM of DTT), and analyzed theoligomeric state of the receptor by SDS-PAGE under non-reducingconditions. R3Mab significantly accelerated the appearance of a ˜25 kDaband representing monomeric FGFR3^(S249C) at the expense of the ˜50 kDadimer, as compared with mock or antibody controls (FIG. 12C); indeed, by2 hr the decrease in dimers was substantially more complete in thepresence of R3Mab. These results indicate that R3Mab shifts theequilibrium between the monomeric and dimeric states of FGFR3^(S249C) infavor of the monomer.

R3Mab does not Promote FGFR3 Down-Regulation

We examined the effect of R3Mab (clone 184.6.1) and anti-FGFR3 hybridomaantibodies on FGFR3 downregulation by analyzing FGFR3 internalizationand degradation in FGFR3 antibody-treated cells. Bladder cancer celllines expressing wild type FGFR3 (RT112) or mutated FGFR3 (S249C inTCC97-7) were treated with R3Mab or hybridoma antibodies 1G6 or 6G1 for4 to 24 hours, then cell lysates were harvested for western blotanalysis of total FGFR3 levels. Treatment with R3Mab did not reduceFGFR3 levels, while treatment with hybridoma mabs 1G6 and 6G1significantly reduced FGFR3 levels. These results suggested that R3Mabdid not promote FGFR3 down-regulation while mabs 1G6 and 6G1 did promoteFGFR3 receptor internalization and down regulation. In a separateexperiment, surface FGFR3 levels were examined using FACS analysis.After 24 hours of R3Mab (clone 184.6.1) treatment of UMUC-14 cells(containing FGFR3 S249C mutation), cell surface FGFR3 levels slightlyincreased. These results demonstrate that R3Mab treatment did notpromote FGFR3 down-regulation.

R3Mab Inhibits Growth and FGFR3 Signaling in Multiple Tumor Models

Next, we examined the effect of R3Mab on the growth of bladder cancercells in vivo. We injected nu/nu mice with RT112 cells (which express WTFGFR3), allowed tumors to grow to a mean volume of ˜150 mm³, and dosedthe animals twice weekly with vehicle or R3Mab. Compared with vehiclecontrol at day 27, R3Mab treatment at 5 or 50 mg/kg suppressed tumorgrowth by about 41% or 73% respectively (FIG. 13A). Analysis of tumorlysates collected 48 hr or 72 hr after treatment showed that R3Mabmarkedly decreased the level of phosphorylated FRS2α (FIG. 13B).Intriguingly, total FRS2α protein levels were also lower inR3Mab-treated tumors, suggesting that FGFR3 inhibition may further leadto downregulation of FRS2α. R3Mab also lowered the amount ofphosphorylated MAPK in tumors, without affecting total MAPK levels (FIG.13B). Thus, R3Mab inhibits growth of RT112 tumor xenografts inconjunction with blocking signaling by WT FGFR3.

We next investigated the effect of R3Mab on growth of xenograftsexpressing mutant FGFR3. R3Mab treatment profoundly attenuated theprogression of Ba/F3-FGFR3^(S249C) tumors (FIG. 13C). Moreover, R3Mabsignificantly inhibited growth of UMUC-14 bladder carcinoma xenografts(FIG. 13D). To evaluate whether R3Mab impacts FGFR3^(S249C) activationin vivo, we assessed the level of FGFR3^(S249C) dimer in tumor lysatescollected 24 hr or 72 hr after treatment. Under non-reducing conditions,the amount of FGFR3^(S249C) dimer was substantially lower in R3Mabtreated tumors as compared with control group, whereas totalFGFR3^(S249C) levels, as judged by the amount detected under reducingconditions, showed little change (FIG. 13E). No apparent accumulation ofFGFR3^(S249C) monomer was observed in tumor lysates, in contrast to theresults in cell culture (FIG. 13E vs. 12A). This could be due to theweak detection sensitivity for monomeric FGFR3 under non-reducingconditions by the rabbit polyclonal anti-FGFR3 antibody used in thisstudy (FIG. 21). Importantly, R3Mab also significantly inhibited thephosphorylation and activation of MAPK in UMUC-14 tumors (FIG. 13E),suggesting that R3Mab inhibits the activity of FGFR3^(S249C) in vivo. Wedid not observe any significant weight loss or other gross abnormalitiesin any the in vivo studies. Furthermore, in a safety study conducted inmice, R3Mab, which binds with similar affinity to both human and murineFGFR3, did not exert any discernable toxicity in any organs, includingbladder (data not shown). Together, these data indicate that multipleexposures to R3Mab are well tolerated in mouse.

Anti-Tumor Activity of R3Mab in Multiple Myeloma Xenograft ModelsInvolves ADCC

To assess whether R3Mab might harbor therapeutic potential for multiplemyeloma, we first tested the effect of R3Mab on the proliferation andsurvival of three t(4; 14)+ cell lines in culture. UTMC-2 cells carry WTFGFR3, while OPM2 and KMS11 harbor a K650E and Y373C substitution,respectively (7). In culture, R3Mab abrogated FGF9-induced proliferationof UTMC-2 cells completely (FIG. 22A). R3Mab modestly inhibited thegrowth of OPM2 cells, but had no apparent effect on the proliferation ofKMS11 cells (FIG. 22 B, C). Since UTMC-2 cells do not form tumors inmice, we evaluated the efficacy of R3Mab against OPM2 and KMS11 tumors.R3Mab almost completely abolished xenograft tumor growth of both celllines (FIG. 14 A, B).

The marked difference in activity of R3Mab against OPM2 and KMS11 tumorcells in vitro and in vivo suggested the possibility that R3Mab may becapable of supporting Fc-mediated immune effector functions againstthese FGFR3-overexpressing tumors. Both cell lines express high levelsof CD55 and CD59 (data not shown), two inhibitors of the complementpathway; accordingly, no complement-dependent cytotoxicity was observed(data not shown). We then focused on ADCC. ADCC occurs when an antibodybinds to its antigen on a target cell, and via its Fc region, engagesFcγ receptors (FcγRs) expressed on immune effector cells (38). To testADCC in vitro, we incubated KMS11 or OPM2 cells with freshly isolatedhuman peripheral blood mononuclear cells (PBMC) in the presence of R3Mabor control antibody. R3Mab mediated significant PBMC cytolytic activityagainst both myeloma cell lines (FIG. 14 C, D). By contrast, R3Mab didnot support cytolysis of bladder cancer RT112 or UMUC-14 cells (FIG. 14E, F). As measured by Scatchard analysis, the multiple myeloma cellsexpress substantially more cell-surface FGFR3 than the bladder carcinomacell lines (˜5-6 fold more receptors per cell; FIG. 23 A, B).

To address the contribution of ADCC to the activity of R3Mab in vivo, weintroduced the previously characterized D265A/N297A (DANA) mutation intothe antibody's Fc domain. This dual substitution in the Fc domain of anantibody abolishes its binding to FcγRs (39), preventing recruitment ofimmune effector cells. The DANA mutation did not alter R3Mab binding toFGFR3 or inhibition of FGFR3 activity in vitro, nor did it change thepharmacokinetics of R3Mab in mice (data not shown); however, itsubstantially abolished in vivo activity against OPM2 or KMS11xenografts (FIG. 14 G, H). By contrast, the DANA mutation did not alterthe anti-tumor activity of R3Mab towards RT112 and UMUC-14 bladdercancer xenografts (FIG. 24 A, B). Together, these results suggest thatFc-dependent ADCC plays an important role in the efficacy of R3Mabagainst OPM2 and KMS11 multiple myeloma xenografts.

Additional Xenograft Studies

R3Mab (clone 184.6.1N54S) was further characterized as follows:

-   -   (a) R3Mab was tested for in vivo efficacy using a tumor        xenograft model based on a liver cancer cell line (Huh7). When        tested at an antibody concentration of 5 mg/kg and 30 mg/kg,        R3Mab significantly inhibited tumor growth in vivo. Tumor growth        was inhibited about 50% compared to tumor growth in control        animals.    -   (b) R3Mab was tested for in vivo efficacy using a tumor        xenograft model based on a breast cancer cell line (Cal-51)        which expressed FGFR3. Results from this efficacy study showed        that the R3Mab antibody was capable of inhibiting tumors in vivo        when tested at antibody concentration range of about 1 mg/kg to        100 mg/kgs. Tumor growth was inhibited about 30% compared to        tumor growth in control animals.

Discussion

The association of FGFR3 overexpression with poor prognosis in t(4; 14)+multiple myeloma patients and the transforming activity of activatedFGFR3 in several experimental models have established FGFR3 as animportant oncogenic driver and hence a potential therapeutic target inthis hematologic malignancy. By contrast, despite reports of a highfrequency of mutation and/or overexpression of FGFR3 in bladdercarcinoma (24, 25, 40), a critical role for FGFR3 signaling in thisepithelial malignancy has not been established in vivo. Moreover, thetherapeutic potential of FGFR3 inhibition in bladder cancer has yet tobe defined. Here we show that genetic or pharmacological interventionwith FGFR3 inhibits growth of several human bladder cancer xenografts inmice. These results demonstrate that FGFR3 function is critical fortumor growth in this setting, underscoring the potential importance ofthis receptor as an oncogenic driver and therapeutic target in bladdercancer. Blockade of FGFR3 function inhibited growth of xenograftsexpressing either WT or mutant FGFR3 alike, suggesting that both formsof the receptor may contribute significantly to bladder tumorprogression. Albeit much less frequently than in bladder cancer, FGFR3mutations or overexpression have been identified in other solid tumormalignancies, including cervical carcinoma (40), hepatocellularcarcinoma (41) and non-small cell lung cancer (42, 43), suggesting apotential contribution of FGFR3 to additional types of epithelialcancer.

The apparent involvement of FGFR3 in diverse malignancies identifiesthis receptor as an intriguing candidate for targeted therapy. Whilesmall molecule compounds that can inhibit FGFR3 kinase activity havebeen described (18-22, 44), the close homology of the kinase domainswithin the FGFR family has hampered the development of FGFR3-selectiveinhibitors. The lack of selectivity of the reported inhibitors makes itdifficult to discern the relative contribution of FGFR3 to the biologyof specific cancer types; further, it may carry safety liabilities,capping maximal dose levels and thus limiting optimal inhibition ofFGFR3. Therefore, to achieve selective and specific targeting of FGFR3,we turned to an antibody-based strategy. We reasoned that an optimaltherapeutic antibody should be capable of blocking not only the WT butalso the prevailing cancer-linked mutants of FGFR3. Furthermore, giventhat dimerization of FGFR3 is critical for its activation, an antibodythat not only blocks ligand binding but also interferes with receptordimerization could be superior. Additional desirable properties wouldinclude the ability to support Fc-mediated effector function and thelong serum half-life conferred by the natural framework of a full-lengthantibody. We focused our screening and engineering efforts to identifyan antibody molecule that combines all of these features, leading to thegeneration of R3Mab. Binding studies demonstrated the ability of R3Mabto compete with FGF ligands for interaction with both the IIIb and IIIcisoforms of FGFR3. Further experiments with transfected BaF/3 cell linesconfirmed the remarkable ability of R3Mab to block both WT and prevalentcancer-associated FGFR3 mutants. In addition, R3Mab exerted significantanti-tumor activity in several xenograft models of bladder cancerexpressing either WT FGFR3 or FGFR3^(S249C), which is the most commonmutant of the receptor in this disease. Pharmacodynamic studiessuggested that the anti-tumor activity R3Mab in these models is based oninhibition of FGFR3 signaling, evident by diminished phosphorylation ofits downstream mediators FRS2α and MAPK. These data further reinforcethe conclusion that FGFR3 is required for bladder tumor progression, asdemonstrated by our FGFR3 shRNA studies.

FGFR3 mutations in bladder cancer represent one of the most frequentoncogenic alterations of a protein kinase in solid tumor malignancies,reminiscent of the common mutation of B-Raf in melanoma (45). Most ofthe activating mutations in FGFR3 give rise to an unpaired cysteine,leading to ligand-independent receptor dimerization and to variousdegrees of constitutive activation. A previous study using a monovalentanti-FGFR3 Fab fragment indicated differential inhibitory activityagainst specific FGFR3 mutants (46); however, the molecular basis forthis variable effect was not investigated. Compared with monovalentantibody fragments, bivalent antibodies have the capacity to induce theclustering of antigens, and in the case of receptor tyrosine kinases,may cause receptor oligomerization and activation. Despite itsfull-length, bivalent configuration, R3Mab displayed universalinhibition of WT FGFR3 and of a wide spectrum of FGFR3 mutants,including variants that are ligand-dependent (FGFR3^(G372C),FGFR3^(Y375C)), constitutively active (FGFR3^(R248C), FGFR3^(S249C)), orboth (FGFR3^(K652E)). These results raise the question: How does R3Mabantagonize both WT and various FGFR3 mutants, including disulfide-linkedvariants?

Based on sequence alignment with FGFR1, the peptide epitope recognizedby R3Mab overlaps with FGFR3 residues involved in binding to ligand andheparin, as well as receptor dimerization. This conclusion was confirmedby crystallographic studies of the complex between R3Mab and theextracellular regions of FGFR3. The X-ray structure revealed that theantibody binds to regions of IgD2 and IgD3 that are critical forligand-receptor interaction as well as receptor-receptor contact. Thus,R3Mab may block WT FGFR3 both by competing for ligand binding and bypreventing receptor dimerization. R3Mab may employ a similar mechanismto inhibit FGFR3^(K652E), which has low constitutive activity, butrequires ligand for full activation. Furthermore, R3Mab binding changesthe relative orientation of FGFR IgD3 with respect to IgD2. This findingraises the formal possibility that the antibody might also inhibitreceptor activation by forcing a conformation that is not conducive tosignal transduction—a notion that requires further study.

To gain better insight into how R3Mab blocks FGFR3 variants possessingan unpaired cysteine, we analyzed the most common mutant, FGFR3^(S249C),in greater detail. Experiments with the free-sulfhydryl blocker DTNBindicated a dynamic equilibrium between the monomeric and dimeric stateof FGFR3^(S249C). Similar equilibrium between oxidized and reducedstates modulated by endogenous redox regulators has been reported forNMDA receptors (46). Incubation of bladder cancer cells expressingFGFR3^(S249C) with R3Mab led to a decline in the amount of receptordimers and a concomitant increase in the level of monomers. Moreover,the purified IgD2-D3 fragment of FGFR3^(S249C) formed dimers insolution; when incubated with R3Mab, the dimers steadily disappearedwhile monomeric FGFR3^(S249C) accumulated. Taken together with thestructural analysis, these results suggest that R3Mab captures monomericFGFR3^(S249C) and hinders its dimerization. Over time, R3Mab shifts theequilibrium towards the monomeric state, blocking constitutive receptoractivity. This mechanism might also explain how R3Mab inhibits othercysteine mutants of FGFR3.

Another important finding of this study was the potent anti-tumoractivity of R3Mab against the t(4; 14)+ multiple myeloma cell lines OPM2and KMS11 in vivo. By contrast, R3Mab had modest to minimal impact onproliferation or survival of these cells in culture. OPM2 and KMS11cells express relatively high cell surface levels of FGFR3 (5-6 foldhigher than RT112 and UMUC-14 bladder carcinoma cells). These higherantigen densities may permit R3Mab to support efficient recruitment ofFcγR-bearing immune effector cells and activation of ADCC. Indeed, inthe presence of human PBMC, R3Mab mediated cytolysis of OPM2 and KMS11cells, but not RT112 or UMUC-14 bladder cancer cells. Moreover, the DANAmutant version of R3Mab, which is incapable of FcγR binding, had noeffect on KMS11 or OPM2 growth in vivo, but still suppressed growth ofRT112 and UMUC-14 tumors similarly to R3Mab. Together, these dataindicate that R3Mab has a dual mechanism of anti-tumor activity: (a) Incells expressing lower surface levels of WT or mutant FGFR3, it blocksligand-dependent or constitutive signaling; (b) In cells expressingrelatively high surface FGFR3 levels, it induces ADCC.

Our results also raise some new questions. First, it is unknown why thebladder cancer cell lines tested in this study display variablesensitivity to R3Mab. Such differential response, which is common fortargeted therapy, may be a reflection of the distinct genetic make-up ofindividual tumors. Indeed, Her2-positive breast cancer cells showvariable sensitivity to anti-Her2 antibody (48), as do various cancercells in response to anti-EGFR antibody (49). In this context,development of additional in vivo models for bladder cancer with WT andmutant FGFR3 is urgently needed to assess sensitivity to FGFR3 moleculesin animals. Moreover, elucidation of predictive biomarkers may helpidentify patients who can optimally benefit from FGFR3-targeted therapy.Secondly, because R3Mab did not induce tumor regression in the models weexamined, future studies should explore whether R3Mab can cooperate withestablished therapeutic agents.

In conclusion, our findings implicate both WT and mutant FGFR3 asimportant for bladder cancer growth, thus expanding the in vivooncogenic involvement of this receptor from hematologic to epithelialmalignancy. Furthermore, our results demonstrate that both WT and mutantFGFR3 can be effectively targeted in tumors with a full-length antibodythat combines the ability to block ligand binding, receptor dimerizationand signaling, as well as to promote tumor cell lysis by ADCC. Theseresults provide a strong rationale for investigating antibody-based,FGFR3-targeted therapies in diverse malignancies associated with thisreceptor.

PARTIAL REFERENCE LIST

-   1. Eswarakumar, V. P., Lax, I., and Schlessinger, J. 2005. Cellular    signaling by fibroblast growth factor receptors. Cytokine Growth    Factor Rev 16:139-149.-   2. L'Hote, C. G., and Knowles, M. A. 2005. Cell responses to FGFR3    signalling: growth, differentiation and apoptosis. Exp Cell Res    304:417-431.-   3. Dailey, L., Ambrosetti, D., Mansukhani, A., and    Basilico, C. 2005. Mechanisms underlying differential responses to    FGF signaling. Cytokine Growth Factor Rev 16:233-247.-   4. Mohammadi, M., Olsen, S. K., and Ibrahimi, O. A. 2005. Structural    basis for fibroblast growth factor receptor activation. Cytokine    Growth Factor Rev 16:107-137.-   5. Grose, R., and Dickson, C. 2005. Fibroblast growth factor    signaling in tumorigenesis. Cytokine Growth Factor Rev 16:179-186.-   6. Chang, H., Stewart, A. K., Qi, X. Y., Li, Z. H., Yi, Q. L., and    Trudel, S. 2005. Immunohistochemistry accurately predicts FGFR3    aberrant expression and t(4; 14) in multiple myeloma. Blood    106:353-355.-   7. Chesi, M., Nardini, E., Brents, L. A., Schrock, E., Ried, T.,    Kuehl, W. M., and Bergsagel, P. L. 1997. Frequent translocation    t(4; 14) (p16.3;q32.3) in multiple myeloma is associated with    increased expression and activating mutations of fibroblast growth    factor receptor 3. Nat Genet 16:260-264.-   8. Fonseca, R., Blood, E., Rue, M., Harrington, D., Oken, M. M.,    Kyle, R. A., Dewald, G. W., Van Ness, B., Van Wier, S. A.,    Henderson, K. J., et al. 2003. Clinical and biologic implications of    recurrent genomic aberrations in myeloma. Blood 101:4569-4575.-   9. Moreau, P., Facon, T., Leleu, X., Morineau, N., Huyghe, P.,    Harousseau, J. L., Bataille, R., and Avet-Loiseau, H. 2002.    Recurrent 14q32 translocations determine the prognosis of multiple    myeloma, especially in patients receiving intensive chemotherapy.    Blood 100:1579-1583.-   10. Pollett, J. B., Trudel, S., Stern, D., Li, Z. H., and    Stewart, A. K. 2002. Overexpression of the myeloma-associated    oncogene fibroblast growth factor receptor 3 confers dexamethasone    resistance. Blood 100:3819-3821.-   11. Bernard-Pierrot, I., Brams, A., Dunois-Larde, C., Caillault, A.,    Diez de Medina, S. G., Cappellen, D., Graff, G., Thiery, J. P.,    Chopin, D., Ricol, D., et al. 2006. Oncogenic properties of the    mutated forms of fibroblast growth factor receptor 3b.    Carcinogenesis 27:740-747.-   12. Agazie, Y. M., Movilla, N., Ischenko, I., and    Hayman, M. J. 2003. The phosphotyrosine phosphatase SHP2 is a    critical mediator of transformation induced by the oncogenic    fibroblast growth factor receptor 3. Oncogene 22:6909-6918.-   13. Ronchetti, D., Greco, A., Compasso, S., Colombo, G., Dell'Era,    P., Otsuki, T., Lombardi, L., and Neri, A. 2001. Deregulated FGFR3    mutants in multiple myeloma cell lines with t(4; 14): comparative    analysis of Y373C, K650E and the novel G384D mutations. Oncogene    20:3553-3562.-   14. Chesi, M., Brents, L. A., Ely, S. A., Bais, C., Robbiani, D. F.,    Mesri, E. A., Kuehl, W. M., and Bergsagel, P. L. 2001. Activated    fibroblast growth factor receptor 3 is an oncogene that contributes    to tumor progression in multiple myeloma. Blood 97:729-736.-   15. Plowright, E. E., Li, Z., Bergsagel, P. L., Chesi, M.,    Barber, D. L., Branch, D. R., Hawley, R. G., and    Stewart, A. K. 2000. Ectopic expression of fibroblast growth factor    receptor 3 promotes myeloma cell proliferation and prevents    apoptosis. Blood 95:992-998.-   16. Chen, J., Williams, I. R., Lee, B. H., Duclos, N., Huntly, B.    J., Donoghue, D. J., and Gilliland, D. G. 2005. Constitutively    activated FGFR3 mutants signal through PLCgamma-dependent and    -independent pathways for hematopoietic transformation. Blood    106:328-337.-   17. Li, Z., Zhu, Y. X., Plowright, E. E., Bergsagel, P. L., Chesi,    M., Patterson, B., Hawley, T. S., Hawley, R. G., and    Stewart, A. K. 2001. The myeloma-associated oncogene fibroblast    growth factor receptor 3 is transforming in hematopoietic cells.    Blood 97:2413-2419.-   18. Trudel, S., Ely, S., Farooqi, Y., Affer, M., Robbiani, D. F.,    Chesi, M., and Bergsagel, P. L. 2004 Inhibition of fibroblast growth    factor receptor 3 induces differentiation and apoptosis in t(4; 14)    myeloma. Blood 103:3521-3528.-   19. Trudel, S., Li, Z. H., Wei, E., Wiesmann, M., Chang, H., Chen,    C., Reece, D., Heise, C., and Stewart, A. K. 2005. CHIR-258, a    novel, multitargeted tyrosine kinase inhibitor for the potential    treatment of t(4; 14) multiple myeloma. Blood 105:2941-2948.-   20. Chen, J., Lee, B. H., Williams, I. R., Kutok, J. L.,    Mitsiades, C. S., Duclos, N., Cohen, S., Adelsperger, J., Okabe, R.,    Coburn, A., et al. 2005. FGFR3 as a therapeutic target of the small    molecule inhibitor PKC412 in hematopoietic malignancies. Oncogene    24:8259-8267.-   21. Paterson, J. L., Li, Z., Wen, X. Y., Masih-Khan, E., Chang, H.,    Pollett, J. B., Trudel, S., and Stewart, A. K. 2004. Preclinical    studies of fibroblast growth factor receptor 3 as a therapeutic    target in multiple myeloma. Br J Haematol 124:595-603.-   22. Grand, E. K., Chase, A. J., Heath, C., Rahemtulla, A., and    Cross, N. C. 2004. Targeting FGFR3 in multiple myeloma: inhibition    of t(4; 14)-positive cells by SU5402 and PD173074. Leukemia    18:962-966.-   23. Gomez-Roman, J. J., Saenz, P., Molina, M., Cuevas Gonzalez, J.,    Escuredo, K., Santa Cruz, S., Junquera, C., Simon, L., Martinez, A.,    Gutierrez Banos, J. L., et al. 2005. Fibroblast growth factor    receptor 3 is overexpressed in urinary tract carcinomas and    modulates the neoplastic cell growth. Clin Cancer Res 11:459-465.-   24. Tomlinson, D. C., Baldo, O., Hamden, P., and    Knowles, M. A. 2007. FGFR3 protein expression and its relationship    to mutation status and prognostic variables in bladder cancer. J    Pathol 213:91-98.-   25. van Rhijn, B. W., Montironi, R., Zwarthoff, E. C., Jobsis, A.    C., and van der Kwast, T. H. 2002. Frequent FGFR3 mutations in    urothelial papilloma. J Pathol 198:245-251.-   26. Tomlinson, D. C., Hurst, C. D., and Knowles, M. A. 2007.    Knockdown by shRNA identifies S249C mutant FGFR3 as a potential    therapeutic target in bladder cancer. Oncogene 26:5889-5899.-   27. Martinez-Torrecuadrada, J., Cifuentes, G., Lopez-Serra, P.,    Saenz, P., Martinez, A., and Casal, J. I. 2005. Targeting the    extracellular domain of fibroblast growth factor receptor 3 with    human single-chain Fv antibodies inhibits bladder carcinoma cell    line proliferation. Clin Cancer Res 11:6280-6290.-   28. Martinez-Torrecuadrada, J. L., Cheung, L. H., Lopez-Serra, P.,    Barderas, R., Canamero, M., Ferreiro, S., Rosenblum, M. G., and    Casal, J. I. 2008. Antitumor activity of fibroblast growth factor    receptor 3-specific immunotoxins in a xenograft mouse model of    bladder carcinoma is mediated by apoptosis. Mol Cancer Ther    7:862-873.-   29. Ornitz, D. M., and Leder, P. 1992. Ligand specificity and    heparin dependence of fibroblast growth factor receptors 1 and 3. J    Biol Chem 267:16305-16311.-   30. d'Avis, P. Y., Robertson, S. C., Meyer, A. N., Bardwell, W. M.,    Webster, M. K., and Donoghue, D. J. 1998. Constitutive activation of    fibroblast growth factor receptor 3 by mutations responsible for the    lethal skeletal dysplasia thanatophoric dysplasia type I. Cell    Growth Differ 9:71-78.-   31. Adar, R., Monsonego-Ornan, E., David, P., and Yayon, A. 2002.    Differential activation of cysteine-substitution mutants of    fibroblast growth factor receptor 3 is determined by cysteine    localization. J Bone Miner Res 17:860-868.-   32. Knowles, M. A. 2008. Novel therapeutic targets in bladder    cancer: mutation and expression of FGF receptors. Future Oncol    4:71-83.-   33. Naski, M. C., Wang, Q., Xu, J., and Ornitz, D. M. 1996. Graded    activation of fibroblast growth factor receptor 3 by mutations    causing achondroplasia and thanatophoric dysplasia. Nat Genet    13:233-237.-   34. Plotnikov, A. N., Schlessinger, J., Hubbard, S. R., and    Mohammadi, M. 1999. Structural basis for FGF receptor dimerization    and activation. Cell 98:641-650.-   35. Olsen, S. K., Ibrahimi, O. A., Raucci, A., Zhang, F.,    Eliseenkova, A. V., Yayon, A., Basilico, C., Linhardt, R. J.,    Schlessinger, J., and Mohammadi, M. 2004. Insights into the    molecular basis for fibroblast growth factor receptor autoinhibition    and ligand-binding promiscuity. Proc Natl Acad Sci USA 101:935-940.-   36. Jebar, A. H., Hurst, C. D., Tomlinson, D. C., Johnston, C.,    Taylor, C. F., and Knowles, M. A. 2005. FGFR3 and Ras gene mutations    are mutually exclusive genetic events in urothelial cell carcinoma.    Oncogene 24:5218-5225.-   37. Ellman, G. L. 1959. Tissue sulfhydryl groups. Arch Biochem    Biophys 82:70-77.-   38. Adams, G. P., and Weiner, L. M. 2005. Monoclonal antibody    therapy of cancer. Nat Biotechnol 23:1147-1157.-   39. Gong, Q., Ou, Q., Ye, S., Lee, W. P., Cornelius, J., Diehl, L.,    Lin, W. Y., Hu, Z., Lu, Y., Chen, Y., et al. 2005. Importance of    cellular microenvironment and circulatory dynamics in B cell    immunotherapy. J Immunol 174:817-826.-   40. Cappellen, D., De Oliveira, C., Ricol, D., de Medina, S.,    Bourdin, J., Sastre-Garau, X., Chopin, D., Thiery, J. P., and    Radvanyi, F. 1999. Frequent activating mutations of FGFR3 in human    bladder and cervix carcinomas. Nat Genet 23:18-20.-   41. Qiu, W. H., Zhou, B. S., Chu, P. G., Chen, W. G., Chung, C.,    Shih, J., Hwu, P., Yeh, C., Lopez, R., and Yen, Y. 2005.    Over-expression of fibroblast growth factor receptor 3 in human    hepatocellular carcinoma. World J Gastroenterol 11:5266-5272.-   42. Cortese, R., Hartmann, O., Berlin, K., and Eckhardt, F. 2008.    Correlative gene expression and DNA methylation profiling in lung    development nominate new biomarkers in lung cancer. Int J Biochem    Cell Biol 40:1494-1508.-   43. Woenckhaus, M., Klein-Hitpass, L., Grepmeier, U., Merk, J.,    Pfeifer, M., Wild, P., Bettstetter, M., Wuensch, P., Blaszyk, H.,    Hartmann, A., et al. 2006. Smoking and cancer-related gene    expression in bronchial epithelium and non-small-cell lung cancers.    J Pathol 210:192-204.-   44. Xin, X., Abrams, T. J., Hollenbach, P. W., Rendahl, K. G., Tang,    Y., Oei, Y. A., Embry, M. G., Swinarski, D. E., Garrett, E. N.,    Pryer, N. K., et al. 2006. CHIR-258 is efficacious in a newly    developed fibroblast growth factor receptor 3-expressing orthotopic    multiple myeloma model in mice. Clin Cancer Res 12:4908-4915.-   45. Davies, H., Bignell, G. R., Cox, C., Stephens, P., Edkins, S.,    Clegg, S., Teague, J., Woffendin, H., Garnett, M. J., Bottomley, W.,    et al. 2002. Mutations of the BRAF gene in human cancer. Nature    417:949-954.-   46. Trudel, S., Stewart, A. K., Rom, E., Wei, E., Li, Z. H., Kotzer,    S., Chumakov, I., Singer, Y., Chang, H., Liang, S. B., et al. 2006.    The inhibitory anti-FGFR3 antibody, PRO-001, is cytotoxic to    t(4; 14) multiple myeloma cells. Blood 107:4039-4046.-   47. Gozlan, H., and Ben-Ari, Y. 1995. NMDA receptor redox sites: are    they targets for selective neuronal protection? Trends Pharmacol Sci    16:368-374.-   48. Hudziak, R. M., Lewis, G. D., Winget, M., Fendly, B. M.,    Shepard, H. M., and Ullrich, A. 1989. p185HER2 monoclonal antibody    has antiproliferative effects in vitro and sensitizes human breast    tumor cells to tumor necrosis factor. Mol Cell Biol 9:1165-1172.-   49. Masui, H., Kawamoto, T., Sato, J. D., Wolf, B., Sato, G., and    Mendelsohn, J. 1984. Growth inhibition of human tumor cells in    athymic mice by anti-epidermal growth factor receptor monoclonal    antibodies. Cancer Res 44:1002-1007.-   50. Pai, R., Dunlap, D., Qing, J., Mohtashemi, I., Hotzel, K., and    French, D. M. 2008. Inhibition of fibroblast growth factor 19    reduces tumor growth by modulating beta-catenin signaling. Cancer    Res 68:5086-5095.-   51. Pegram, M., Hsu, S., Lewis, G., Pietras, R., Beryt, M.,    Sliwkowski, M., Coombs, D., Baly, D., Kabbinavar, F., and Slamon, D.    1999 Inhibitory effects of combinations of HER-2/neu antibody and    chemotherapeutic agents used for treatment of human breast cancers.    Oncogene 18:2241-2251.-   52. Lee, C. V., Liang, W. C., Dennis, M. S., Eigenbrot, C.,    Sidhu, S. S., and Fuh, G. 2004. High-affinity human antibodies from    phage-displayed synthetic Fab libraries with a single framework    scaffold. J Mol Biol 340:1073-1093.-   53. Liang, W. C., Dennis, M. S., Stawicki, S., Chanthery, Y., Pan,    Q., Chen, Y., Eigenbrot, C., Yin, J., Koch, A. W., Wu, X., et    al. 2007. Function blocking antibodies to neuropilin-1 generated    from a designed human synthetic antibody phage library. J Mol Biol    366:815-829.-   54. Carter, P., Presta, L., Gorman, C. M., Ridgway, J. B., Henner,    D., Wong, W. L., Rowland, A. M., Kotts, C., Carver, M. E., and    Shepard, H. M. 1992. Humanization of an anti-p185HER2 antibody for    human cancer therapy. Proc Natl Acad Sci USA 89:4285-4289.-   55. Sidhu, S. S., Li, B., Chen, Y., Fellouse, F. A., Eigenbrot, C.,    and Fuh, G. 2004. Phage-displayed antibody libraries of synthetic    heavy chain complementarity determining regions. J Mol Biol    338:299-310.-   56. Otwinowski, Z. a. M., W. 1997. Processing of X-ray diffraction    data collected in oscillation mode. Methods in Enzymology    276:307-326.-   57. McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C., and    Read, R. J. 2005. Likelihood-enhanced fast translation functions.    Acta Crystallogr D Biol Crystallogr 61:458-464.-   58. Emsley, P., and Cowtan, K. 2004. Coot: model-building tools for    molecular graphics. Acta Crystallogr D Biol Crystallogr    60:2126-2132.-   59. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. 1997.    Refinement of macromolecular structures by the maximum-likelihood    method. Acta Crystallogr D Biol Crystallogr 53:240-255.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention.

1. A crystal of FGFR3 complexed with an anti-FGFR3 antibody comprising ahuman FGFR3 comprising sequence of SEQ ID NO:272 or conservativesubstitutions thereof complexed with an anti-FGFR3 antibody comprising(a) a light chain variable domain comprising the amino acid sequence ofSEQ ID NO:274 or conservative substitutions thereof, and (b) a heavychain variable domain comprising the amino acid sequence of SEQ IDNO:275 or conservative substitutions thereof.
 2. Crystalline form of acomplex of FGFR3 and an anti-FGFR3 antibody. space group P2₁2₁2₁ withcell parameters of a=58.5 Å, b=99.3 Å and c=143.7 Å.
 3. A crystal of a1:1 complex of FGFR3 and an anti-FGFR3 antibody having a space grouphaving a space group symmetry of P2₁2₁2₁ and comprising a unit cellhaving the dimensions of a, b, and c, wherein a=58.5 Å, b=99.3 Å andc=143.7 Å.
 4. A cocrystal of FGFR3 with an anti-FGFR3 antibody havingthe three-dimensional coordinates of Table
 6. 5. The crystal of claim 4,wherein the crystal diffracts X-rays for the determination of atomiccoordinates to a resolution of 5 Å or better.
 6. A compositioncomprising a crystal of any of claims 1-5, and a carrier.
 7. A moleculeor molecular complex comprising at least a portion of the binding siteof FGFR3 or conservative substitution thereof, wherein the binding sitecomprises at least one amino acid residue corresponding to residues 158,170, 171, 173, 175, and/or 315 or mixtures thereof, the binding sitedefined by a set of points having a root mean square deviation of lessthan about 0.70 Å from points representing the backbone atoms of theamino acids as represented by the structure coordinates listed in Table6.
 8. The molecule or molecular complex of claim 7, wherein the bindingsite comprises at least one amino acid residue corresponding to residues158, 159, 169, 170, 171, 173, 175, 205, 207, and/or 315 or mixturesthereof.
 9. The molecule or molecular complex of claim 7, wherein thebinding site comprises at least one amino acid residue corresponding toresidues 154, 155, 158, 159, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 177, 202, 205, 207, 210, 212, 214,216, 217, 241, 246, 247, 248, 278, 279, 280, 281, 282, 283, 314, 315,316, 317 and/or 318 or mixtures thereof.
 10. A computer-implementedmethod for causing a display of a graphical three-dimensionalrepresentation of the structure of a portion of a crystal of FGFR3complexed with an anti-FGFR3 antibody, or structural homologs thereof,wherein the method comprises: causing said display of said graphicalthree-dimensional representation by a computer system programmed withinstructions for transforming structure coordinates into said graphicalthree-dimensional representation of said structure and for displayingsaid graphical three-dimensional representation, wherein said graphicalthree-dimensional representation is generated by transforming saidstructure coordinates into said graphical three-dimensionalrepresentation of said structure, wherein said structure coordinatescomprise structure coordinates of the backbone atoms of the portion ofthe crystal, wherein the portion of the crystal comprises an FGFR3binding site, and wherein the crystal has the space group symmetryP2₁2₁2₁.
 11. The computer-implemented method of claim 10, wherein theFGFR3:anti-FGFR3 antibody crystal comprises a polypeptide comprising anamino acid sequence shown in Table 6 or conservative substitutionthereof, and further comprises an antibody comprising (a) a light chainvariable domain comprising the amino acid sequence of SEQ ID NO:274 orconservative substitutions thereof, and (b) a heavy chain variabledomain comprising the amino acid sequence of SEQ ID NO:275 orconservative substitutions thereof.
 12. The computer-implemented methodof claim 10, wherein the structure coordinates are defined in Table 6.13. The computer-implemented method of claim 10, wherein the structurecoordinates comprise the structure coordinates of the backbone atoms ofthe amino acid residues corresponding to residues 158, 170, 171, 173,175, and/or 315 of FGFR3.
 14. The computer-implemented method of claim10, wherein the structure coordinates comprise the structure coordinatesof the backbone atoms of the amino acid residues corresponding toresidues 154, 155, 158, 159, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 177, 202, 205, 207, 210, 212, 214,216, 217, 241, 246, 247, 248, 278, 279, 280, 281, 282, 283, 314, 315,316, 317, 318 of FGFR3.
 15. The computer-implemented method of claim 10,wherein the structure coordinates are determined by homology modeling.16. A machine-readable data storage medium comprising a data storagematerial encoded with machine-readable instructions for: (a)transforming data into a graphical three-dimensional representation forthe structure of a portion of a crystal of FGFR3 complexed with ananti-FGFR3 antibody, or structural homologs thereof; and (b) causing thedisplay of said graphical three-dimensional representation; wherein saiddata comprise structure coordinates of the backbone atoms of the aminoacids defining an FGFR3 binding site; and wherein the crystal orstructural homolog has the space group symmetry P2₁2₁2₁.
 17. A computersystem for displaying a three-dimensional graphical representation forthe structure of a portion of a crystal of FGFR3 complexed with ananti-FGFR3 antibody, or structural homologs thereof, comprising: (a) amachine-readable data storage medium comprising a data storage materialencoded with machine-readable data, wherein said data comprise structurecoordinates of the backbone atoms of the amino acids defining an FGFR3binding site, wherein the crystal or structural homolog has the spacegroup symmetry P2₁2₁2₁; (b) a working memory; (c) a central processingunit coupled to said working memory and to said machine-readable datastorage medium for processing said machine-readable data into sadthree-dimensional graphical representation; and (d) a display coupled tosaid central processing unit for displaying said three-dimensionalgraphical representation.
 18. A method for obtaining structuralinformation about a molecule or molecular complex comprising applying atleast a portion of the structure coordinates of a FGFR3 complexed withan anti-FGFR3 antibody to an X-ray diffraction pattern of the moleculeor molecular complex's crystal structure to cause the generation of athree-dimensional electron density map of at least a portion of themolecule or molecular complex; wherein the FGFR3:anti-FGFR3 antibodycrystal comprises a polypeptide comprising an amino acid sequence of SEQID NO:272 or conservative substitution thereof, and further comprises anantibody comprising (a) a light chain variable domain comprising theamino acid sequence of SEQ ID NO:274 or conservative substitutionsthereof, and (b) a heavy chain variable domain comprising the amino acidsequence of SEQ ID NO:275 or conservative substitutions thereof; whereinthe FGFR3:anti-FGFR3 antibody crystal diffracts x-rays for thedetermination of atomic coordinates to a resolution of 5 Å or better.19. A method of screening for molecules that may be antagonists oragonists of FGFR3 comprising: (a) computationally screening agentsagainst a three-dimensional model to identify potential antagonists oragonists of FGFR3; wherein the three-dimensional model comprises athree-dimensional model of at least a portion of a crystal of a FGFR3complexed with an anti-FGFR3 antibody; wherein the three dimensionalmodel is generated from at least a portion of the structure coordinatesof the crystal by a computer algorithm for generating athree-dimensional model of the crystal useful for identifying agentsthat are potential antagonists or agonists of FGFR3; wherein the FGFR3:anti-FGFR3 antibody crystal comprises a polypeptide comprising an aminoacid sequence SEQ ID NO:272 or conservative substitution thereof, andfurther comprises an antibody comprising (a) a light chain variabledomain comprising the amino acid sequence of SEQ ID NO:274 orconservative substitutions thereof, and (b) a heavy chain variabledomain comprising the amino acid sequence of SEQ ID NO:275 orconservative substitutions thereof; and wherein the FGFR3:anti-FGFR3antibody crystal diffracts x-rays for the determination of atomiccoordinates to a resolution of 5 Å or better.